Polypeptides and uses thereof for treatment of autoimmune disorders and infection

ABSTRACT

This invention relates to C1ORF32 protein and its variants and fragments and fusion proteins thereof, pharmaceutical composition comprising same and methods of use therof for treatment of immune related disorders and infections.

FIELD OF THE INVENTION

The present invention relates to C1ORF32 protein, and its variants,fragments and fusion proteins thereof, pharmaceutical compositionscomprising same and methods of use thereof for treatment of immunerelated disorders and infections.

BACKGROUND OF THE INVENTION

The balance between proinflammatory mechanisms and the dampening ofexcessive immune activation is important for treatment of infectiousdisease and also for treatment of autoimmune disease. The formerbenefits from an activated immune system while the latter requiresreduce immune system activity. Thus, the immune system has thereciprocal tasks to protect the host against invading pathogens, butsimultaneously to prevent damage resulting from unwanted reactions toself antigens.

The latter part is known as immune tolerance and performed by a complexset of interactive and complementary pathways, which regulate immuneresponses. T cells have the ability to react to a variety of antigens,both self and nonself. Therefore, there are many mechanisms that existnaturally to eliminate potentially self-reactive responses—this is knownas natural tolerance. The main mechanism for eliminating potentialauto-reactive T cells occurs in the thymus and is known as centraltolerance. Some potentially autoreactive T cells escape centraltolerance and, therefore, peripheral tolerance mechanisms also exist.Despite these mechanisms, some self-reactive T cells may ‘escape’ and bepresent in the repertoire; it is believed that their activation may leadto autoimmune disease.

Studies on therapeutic tolerance have attempted to induce and amplifypotent physiological mechanisms of tolerance in order to eliminate orneutralize self-reactive T cells and prevent or treat autoimmunediseases. One way to induce tolerance is by manipulation of theinteraction between costimulatory ligands and receptors on antigenpresenting cells (APCs) and lymphocytes.

CTLA-4 is the most extensively studied costimulatory molecule whichdown-regulates immune responses. The attributes of immunosuppressivequalities and capacity to induce tolerance have made its recognition asa potential immuno-therapeutic agent for autoimmune mediatedinflammatory disorders. Abatacept (commercial name: Orencia) is a fusionprotein composed of the ECD (extracellular domain) of CTLA-4 fused tothe Fc fragment of hIgG1. Abatacept is believed to induce costimulationblockade, which has been approved for treating patients with rheumatoidarthritis, by effectively interfering with the inflammatory cascade.

Induction of disease control with the current therapies, followed byprogressive withdrawal in parallel with re-establishing immunetolerance, may be an attractive approach in the future of autoimmunetherapies. Furthermore, due to their immune specificity, in the absenceof global immunosuppression, such therapies should be safer.

T helper type 1 (Th1) cells are induced by IL-12 and produce IFN-γ,while T helper type 2 (Th2) cells secrete IL-4, IL-5 and IL-13. Th1cells can mediate proinflammatory or cell-mediated immune responses,whereas Th2 cells mainly promote certain types of humoral immunity. Someimmune related diseases, such as autoimmune reactions, inflammation, andinfection, are characterized by a dysregulation of the pro-versusanti-inflammatory tendencies of the immune system, as well as animbalance in the Th1 versus Th2 cytokine balance. During inflammation,induction of a shift in the balance from Th1 to Th2 protects theorganism from systemic ‘overshooting’ with Th1/pro-inflammatorycytokines, by reducing the inflammatory tendencies of the immune systemImmunomodulatory therapies that are associated with a Th1 to Th2 immuneshift have protective effects in Th1-mediated autoimmune diseases, suchas multiple sclerosis and rheumatoid arthritis. For example, Laquinimod,which has demonstrated efficacy in animal models of several autoimmunediseases including MS, shows immunomodulatory effects through Th1/Th2shift, and does not lead to immunosuppression. Glatiramer acetate(Copaxone) also induces Th1/Th2 shift with decreased secretion ofproinflammatory cytokines, and increased secretion of antiinflammatorycytokines. Furtheremore, GA-specific Th2 cells are able to migrateacross the blood-brain barrier and cause in situ bystander suppressionof autoaggressive Th1 T cells.

The balance between proinflammatory mechanisms and the dampening ofexcessive immune activation is also critical for successful clearance ofa pathogen without harm to the host. Excessive immune activation maylead to autoimmune attacks, while too little immune activation will notresult in clearance of the pathogen from the host. Chronic pathogensexploit co-inhibitory pathways to attenuate Ag-specific T cell immunity.Emerging data from a wide range of studies on acute and chronicinfections support an important role for negative costimulatoryreceptors in controlling infection. Most notably, exhausted T cells,functionally impaired T cells which are present during chronic infectionand are characterized by reduced proliferative and cytotoxic abilities,express high levels of multiple co-inhibitory receptors such as CTLA-4,PD-1, and LAGS (Crawford et al., Curr Opin Immunol. 2009; 21:179-186;Kaufmann et al., J Immunol 2009; 182:5891-5897, Sharpe et al., NatImmunol 2007; 8:239-245). Furthermore, the exhausted phenotype can bereversed by blocking co-inhibitory pathways (Rivas et al., J Immunol.2009; 183:4284-91; Golden-Mason et al., J Virol. 2009; 83:9122-30), thusallowing restoration of anti viral immune function, supportingtherapeutic application of co-inhibitory blockade in viral infection.

One potentially promising strategy to control chronic infections such ashuman immunodeficiency virus, hepatitis B virus, and hepatitis C virusis therapeutic vaccination, which aims to reduce persisting virus bystimulating a patient's own antiviral immune responses.

However, this approach has fallen short of expectations, becauseantiviral T cells generated during chronic infections often becomefunctionally exhausted, as explained above, and thus do not respondproperly to therapeutic vaccination. Therefore, it is necessary torestore T cell effector functions and effectively boost endogenousT-cell responses in order to develop therapeutic vaccines againstchronic viral infections. Blocking the negative signaling pathways, PD-1and CTLA-4, could restore the host immune system, enabling it to respondto further stimulation. Blockade of the PD-1/PD-L1 pathway, for example,is able to restore functional capabilities to exhausted CTLs (Hofmeyeret al, J. Biomed. & Biotech. Vol. 2011, Art. ID 451694). Combiningtherapeutic vaccination along with the blockade of inhibitory signalscould synergistically enhance functional CD8(+) T-cell responses andimprove viral control in chronically infected individuals, providing apromising strategy for the treatment of chronic viral infections. (Ha etal, Immunol Rev. 2008 June; 223:317-33). Antibodies to PD-1 and CTLA-4are currently in clinical trials in chronic hepatitis C, as promisingcandidates for combination with both prophylactic and therapeuticvaccines (Diepolder and Obst, Expert Rev Vaccines. 2010 March;9(3):243-7).

The therapeutic potential of co-inhibition blockade for treating viralinfection was extensively studied by blocking the PD-1/PD-L1. Blockingthis pathway was shown to be efficacious in several animal models ofinfection including acute and chronic Simian immunodeficiency virus(SIV) infection in rhesus macaques (Valu et al., Nature 2009;458:206-210) and in mouse model of chronic viral infection withlymphocytic choriomeningitis virus (LCMV) (Barber et al., Nature. 2006;439:682-7).

Modulation of costimulatory pathway has also been proven effective inoptimizing antiviral immunity by limiting the memory T cell response toits protective capacities (Teijaro et al., J Immunol. 2009: 182;5430-5438). This has been demonstrated in models of influenza infectionin which inhibiting CD28 costimulation with CTLA4-Ig suppressed primaryresponses in naive mice infected with influenza, but was remarkablycurative for memory CD4 T cell-mediated secondary responses to influenzaleading to improved clinical outcome and increased survival to influenzachallenge. The curative effect of CTLA4-Ig on secondary responses wasaccompanied by inhibition of proliferation and egress of lymphoid naiveand memory T cells, while leaving lung resident memory CD4 T cellresponses intact thus maintaining enhanced and rapid lung viralclearance mediated by memory CD4 T cells, yet reducing lungimmunopathology.

These data demonstrate an active and reversible role for molecules ofthe B7:CD28 family, PD-1, CTLA-4, and their ligands, in virus-specific Tcell exhaustion associated with chronic viral infection and point topromising potential for immunotherapeutic interventions based onmanipulation of these inhibitory networks.

Regulating costimulation using agonists and antagonists to variouscostimulatory proteins has been extensively studied as a strategy fortreating autoimmune diseases, graft rejection, allergy and cancer. Thisfield has been clinically pioneered by CTLA4-Ig (Abatacept, Orencia®)that is approved for treatment of RA, and by the anti-CTLA4 antibody(Ipilimumab, Yervoy®), recently approved for the treatment of melanoma.Other costimulation regulators are currently in advanced stages ofclinical development including anti PD-1 antibody (MDX-1106) which is indevelopment for treatment of advanced/metastatic clear-cell renal cellcarcinoma (RCC) and anti-CD40L Antibody (BG9588, Antova®) for treatmentof renal allograft transplantation. In addition, such agents are inclinical development for viral infections, for example the anti PD-1 Ab,MDX-1106, is being tested for treatment of hepatitis C. Another exampleis CP-675,206 (tremelimumab) and anti-CTLA4 Ab which is in a clinicaltrial in hepatitis C virus-infected patients with hepatocellularcarcinoma.

B cells play a critical role in recognition of foreign antigens andsubsequent production of antibodies in the specific humoral adaptiveimmune responses that provide protection against various types ofinfectious agents. B cells play a critical role in recognition offoreign antigens and they produce the antibodies necessary to provideprotection against various type of infectious agents. T cell help to Bcells is a pivotal process of adaptive immune responses. Follicularhelper T (Tfh) cells are a subset of CD4+ T cells specialized in B cellhelp (reviewed by Crotty, Annu. Rev. Immunol. 29: 621-663, 2011). Tfhcells express the B cell homing chemokine receptor, CXCR5, which drivesTfh cell migration into B cell follicles within lymph nodes in aCXCL13-dependent manner. The requirement of Tfh cells for B cell helpand T cell-dependent antibody responses, indicates that this cell typeis of great importance for protective immunity against various types ofinfectious agents, as well as for rational vaccine design.

Tfh cells selectively express a wealth of surface proteins, which areinvolved in their selective localization (such as CXCR5) and in directphysical interactions with B cells to provide B cell help. Among thelatter group are several members of the costimulatory proteins familywhich are highly expressed in Tfh cells, including the inducibleco-stimulatory receptor ICOS, and the negative costimulators (inhibitoryreceptors) PD-1 and BTLA (Crotty, Annu. Rev. Immunol. 29: 621-663,2011).

BRIEF SUMMARY OF THE INVENTION

The background art fails to provide therapies that target multiple cellsand pathways involved in autoimmunity and inflammation, such as Th1,Th17, Th22, Th2, Tregs, or other cells that secrete, or influence othercells that secrete, inflammatory molecules such as cytokines,metalloproteases, chemokines and other molecules. The background artalso does not teach such therapies that are targeted with regard toproviding a balance between excessive immune activation and desirablepro-inflammatory immune activation, for the treatment of autoimmunediseases and infectious disease.

The present invention is of C1ORF32 protein, and its variants, fragmentsand fusion proteins thereof, pharmaceutical compositions comprising sameand methods of use therof for treatment of immune related disorders andinfections.

With regard to treatment for immune related diseases, it should be notedthat such diseases may optionally relate to any disease in which it isdesirable to induce immune tolerance. With regard to treatment ofinfections, it should be noted that such diseases may optionally relateto any disease in which T cell exhaustion to a foreign pathogenicantigen plays a role.

According to at least some embodiments of the present invention, thereare provided C1ORF32 polypeptides, as an isolated polypeptide,comprising an amino acid sequence of C1ORF32 IgV domain fragment, setforth in any one of SEQ ID NOs: 29, 30, 41-105, with the proviso thatthe amino acid sequence does not include the complete, exact sequence ofSEQ ID NO: 35. Optionally, the isolated polypeptide has less than 90%identity with SEQ ID NO:35, optionally has less than 85% identity withSEQ ID NO:35 and optionally has less than 80% identity with SEQ IDNO:35. Also optionally and preferably, the isolated polypeptide consistsessentially of the amino acid sequence as set forth in any one of SEQ IDNOs: 29, 30, 41-105, or optionally up to 95% identical thereof. It isfurther contemplated that optionally the C1ORF32 IgV domain fragment maybe extended beyond the border delimited by the end of SEQ ID NO:35, aslong as one or more of the above conditions regarding sequence identityare met. A further description of such fragments is provided with regardto Table 1 below.

Briefly, a sequence alignment and comparison is provided between SEQ IDNO:35 and SEQ ID NO:29, as an example of the above described inventivesequences:

Query: 1 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFKSYCQDRMGESLGMSSTRAQSL 60LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFKSYCQDRMGESLGMSSTRAQSL Sbjct: 1LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFKSYCQDRMGESLGMSSTRAQSL 60 Query:61 SKRNLEWDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRGREITIVHDADLQIGKLMWGDS 120SKRNLEWDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRGREITIVHDADLQIGKLMWGDS Sbjct: 61SKRNLEWDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRGREITIVHDADLQIGKLMWGDS 120 Query:121 GLYYCIITTPDDLEGKNEGSLGLLVLGRTGLLADLLPSFAVEIMPE 166GLYYCIITTPDDLEGKNEDSVELLVLG Sbjct: 121GLYYCIITTPDDLEGKNEDSVELLVLG................... 147

For this non-limiting example, SEQ ID NO:29 is shorter than SEQ ID NO:35but as noted above, for some inventive sequences, the inventive aminoacid sequence is longer than SEQ ID NO:35, but still fulfills one ormore of the above conditions regarding sequence identity. An equivalentdifference exists between the inventive sequences and SEQ ID NO:36, suchthat the inventive sequences are as described above and herein, theproviso that the amino acid sequence does not include the complete,exact sequence of SEQ ID NO: 36. Optionally, the isolated polypeptidehas less than 90% identity with SEQ ID NO:36, optionally has less than85% identity with SEQ ID NO:36 and optionally has less than 80% identitywith SEQ ID NO:36. It is further contemplated that optionally theC1ORF32 IgV domain fragment may be extended beyond the border delimitedby the end of SEQ ID NO:36, as long as one or more of the aboveconditions regarding sequence identity are met.

According to at least some embodiments, there is provided an isolatedpolypeptide comprising a soluble C1ORF32 polypeptide or fragment orvariant thereof, having an amino acid substitution preventing a cleavageof the C1ORF32 ECD (SEQ ID NO:14) between amino acids F and A atpositions 179 and 180 of any of H19011_1_P8_V1 or H19011_1_P8 (Seq IDNOs: 4 or 5). Optionally, the amino acid substitution in the cleavagesite of C1ORF32 ECD (SEQ ID NO:14) at positions 179 and 180 of any ofH19011_1_P8_V1 or H19011_1_P8 (Seq ID NOs: 4 or 5) is selected fromFA->GA; FA->AA; and FA->GG. Optionally and preferably, the polypeptidehas an amino acid sequence as set forth in any one of SEQ ID NOs:45, 64,and 96.

According to at least some embodiments of the present invention, thereare provided C1ORF32 polypeptides, optionally provided as fusionproteins containing a C1ORF32 polypeptide. C1ORF32 fusion polypeptidesoptionally have a first fusion partner comprising part of a C1ORF32soluble polypeptide, or a sequence homologous thereto, and a secondfusion partner composed of a heterologous sequence (respectivelynon-C1ORF32), fused together directly or indirectly via a peptide linkersequence or a chemical linker.

Optionally, the fusion protein comprises the polypeptide as describedherein, fused to a heterologous sequence, directly or indirectly via alinker peptide, a polypeptide sequence or a chemical linker.

Optionally the heterologous sequence comprises at least a portion of animmunoglobulin constant domain. Also optionally, the constant domaincomprises an immunoglobulin heavy chain constant region corresponding toan antibody isotype selected from the group consisting of an IgG1, IgG2,IgG3, IgG4, IgM, IgE, IgA and IgD.

Optionally the immunoglobulin constant domain comprises the hinge, CH2and CH3 regions of a human IgG immunoglobulin, selected from the groupconsisting of Cγ1, Cγ2, Cγ3 and Cγ4 chain.

Optionally, the fusion protein further comprises a domain that mediatesdimerization or multimerization of the fusion protein to formhomodimers, heterodimers, homomultimers, or heteromultimers. Optionally,the domain that mediates dimerization or multimerization is selectedfrom the group consisting of one or more cysteines that are capable offorming an intermolecular disulfide bond with a cysteine on the partnerfusion protein, a coiled-coil domain, an acid patch, a zinc fingerdomain, a calcium hand domain, a CHI region, a CL region, a leucinezipper domain, an SH2 (src homology 2) domain, an SH3 (src Homology 3)domain, a PTB (phosphotyrosine binding) domain, a WW domain, a PDZdomain, a 14-3-3 domain, a WD40 domain, an EH domain, a Lim domain, anisoleucine zipper domain, and a dimerization domain of a receptor dimerpair.

According to at least some embodiments of the present invention, thereis provided a dimeric protein comprising a first and a second fusionprotein, wherein the first and the second fusion proteins comprise thefusion protein as described herein and wherein the first and the secondfusion proteins are bound to one another by covalent or noncovalentbonds to form a dimer.

According to at least some embodiments, the isolated polypeptide is atleast 80, 90, 95, 96, 97, 98 or 99% homologous to a polypeptide setforth in any one of SEQ ID NOs: 29, 30, 41-105. The C1ORF32 polypeptidemay be of any species of origin. In further embodiments, the C1ORF32polypeptide is of murine, non-human primate or human origin.

Without wishing to be limited by a single hypothesis, according to atleast some embodiments the C1ORF32 protein, which may optionallycomprise a fusion protein, inhibits the inflammatory activity of Th1,Th17, Th22, or other cells that secrete, or cause other cells tosecrete, inflammatory molecules, including, but not limited to,IL-1beta, TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6, IL-23, IL-22,IL-21, and MMPs. Again without wishing to be limited by a singlehypothesis, according to at least some embodiments the C1ORF32 proteincan also increase the suppressive capacity of Tregs or theimmunomodulatory activity of Th2 cells. The C1ORF32 fusion protein canalso increase the production of anti-inflammatory molecules such as thecytokine IL-10.

Optionally, the fragment is of at least about 20 amino acids of theextracellular domain of C1ORF32, but no more than 169 amino acids of theextracellular domain, in order to fulfill the conditions regardingsequence identity as described with regard to SEQ ID NO:35 as describedabove. Optionally, the fragment is of at least about 21, 22, 23, 24, 25and so forth amino acids of the extracellular domain of C1ORF32, but nomore than 100, 101, 102 and so forth, up to 168 amino acids of theextracellular domain, as described above, optionally including anyintegral value between 20 and 169 amino acids in length, although asnoted above, the inventive sequence may optionally have more amino acidsthan the ECD of SEQ ID NO:35. Optionally the polypeptide is attached toa detectable or therapeutic moiety.

According to at least some embodiments of the present invention, thereis provided a method for prevention of damage to the myelin coat ofneural cells in the central nervous system in MS (multiple sclerosis)patients comprising administering to a subject in need thereof apharmaceutical composition comprising: a soluble molecule having theC1ORF32 polypeptide, selected from the group consisting of SEQ ID NOs:29, 30, 41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof; optionally provided as a pharmaceutical composition.Optionally, the fragment is as described above.

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinatingdisorder of the central nervous system (CNS), which involves autoimmuneresponses to myelin antigens. It is characterized by lesions within theCNS and demyelination is a key feature of these lesions. Autoreactive Tcells are thought to initiate an autoimmune response directed againstcomponents of CNS myelin. The main targets of the autoimmune reactionsare thought to be myelin basic protein (MBP), proteolipid protein (PLP)and myelin oligodendrocyte glycoprotein (MOG). Experimental autoimmuneencephalomyelitis (EAE), an animal model of MS induced by immunizationwith myelin components in adjuvant, shows comparable neuronal pathology.Without wishing to be limited by a single hypothesis, studies in EAEhave provided convincing evidence that T cells specific forself-antigens mediate pathology in these diseases.

According to at least some embodiments of the present invention, thereis provided an isolated soluble C1ORF32 polypeptide, selected from thegroup consisting of SEQ ID NOs: 29, 30, 41-105, fragment, variant, orhomolog thereof; optionally as a fusion protein or conjugate, whereinsaid polypeptide or said fusion protein or conjugate is used foranti-immune related condition immunotherapy for an immune relateddisorder and/or infection as described herein, optionally provided as apharmaceutical composition.

Optionally treating comprises one or more of preventing, curing,managing, reversing, attenuating, alleviating, minimizing, suppressing,managing, or halting the deleterious effects of the above-describeddiseases.

Optionally, managing comprises reducing the severity of the disease,reducing the frequency of episodes of the disease, reducing the durationof such episodes, or reducing the severity of such episodes or acombination thereof.

In another embodiment, the C1ORF32 polypeptides, fragments or variantsor homologs thereof, fusion proteins or conjugates comprising same, orpharmaceutical composition comprising same, can be used to treatpatients who do not respond to TNF blockers.

According to at least some embodiments, the present invention provides amethod for combining therapeutic vaccination with an antigen along withadministration of the foregoing pharmaceutical composition for treatmentof infection. According to at least some embodiments of the presentinvention, the antigen is a viral antigen, a bacterial antigen, fungalantigen and/or other parasite antigen.

According to at least some embodiments, the present invention furtherprovides a method for combining the foregoing pharmaceuticalcomposition, used as adjuvant, along with an antigen in a vaccine, inorder to increase the immune response. According to at least someembodiments of the present invention, the antigen is a viral antigen, abacterial antigen, fungal antigen, parasite antigen, and/or otherpathogen's antigen.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic drawing of a full length soluble C1ORF32protein. Signal Peptide is shown in italic face font; two potentialstart points of IgV are shown in underlined italic and bold shape; IgVdomain is in bold face font; amino acid region deleted in H19011_1_P9(SEQ ID NO:6) as compared to the H19011_1_P8 (SEQ ID NO:4) is shown indashed underline; the locations of the three SNPs (DSVE->GSLG) is shownin double underlined italic and bold shape; the location of pointmutations (FA->GA, FA->AA, FA->GG) is shown in bold and dashedunderline. Transmembrane domain (TM) of H19011_1_P8 (SEQ ID NO:4) isshown in light font with dotted underline; in H19011_1_P9 (SEQ ID NO:6)the TM starts at amino acid residues “FVG . . . ”, 2 amino acidsdownstream to the start point of the H19011_1_P8 (SEQ ID NO:4)transmembrane domain. Vertical bars represent five possible C-terminalend points of the soluble C1ORF32 proteins from position 1 on the left(end of IgV) to position 5 on the right (end of predicted ECD).

FIG. 2 shows SDS-PAGE results for produced proteins.

FIG. 3 shows inhibitory effect of Fc-fused C1ORF32 ECD (SEQ ID NO:108),Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), and C1ORF32ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) on mouse CD4+ T cell IFNγsecretion. Purified CD4+CD25− T cells, 1×10⁵ per well, were stimulatedwith anti-CD3 mAb (2 ug/mL) in the presence of test proteins or controlmouse IgG2a at 0, 3 and 10 μg/ml. Culture supernatants were collected 48hrs post stimulation and analyzed using mouse IFNγ ELISA kit. Resultsare shown as Mean±SD of four wells per point (Student's t-test,***P<0.001, compared with control mouse IgG2a).

FIG. 4 shows inhibitory effect of Fc-fused C1ORF32 ECD (SEQ ID NO:108),Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), and C1ORF32ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) on expression of the earlyTCR activation marker, CD69. 1×10⁵ CD4+CD25− T cells were stimulatedwith plate bound anti-CD3 (2 μg/ml) together with test proteins orcontrol mIgG2a (10 μg/ml). Cells were analyzed at 48 hrs for theexpression CD69 by flow cytometry (**; P value<0.001 compare to controlmIgG2A, student's T test).

FIG. 5 shows inhibitory effect of Fc-fused C1ORF32 ECD (SEQ ID NO:108),(two different batches produced in CHO—S And in CHO-DG44), Fc-fusedC1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), and C1ORF32 ECD-DeltaRTGLLADLLPSFAVEIM (SEQ ID NO:112) (two different batches produced inCHO—S And in CHO-DG44) on naive T cell proliferation. No inhibition wasobserved with the IgG2a isotype control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in at least some embodiments, relates to any oneof the C1ORF32 proteins, selected from the group consisting of SEQ IDNOs: 29, 30, 41-105, fragments, variants and homologs thereof and fusionproteins and conjugates containing same, and pharmaceutical compositionscomprising same, and nucleic acid sequences encoding same, and the usethereof as a therapeutic agent for treatment of immune related disorderand/or infection, and/or optionally the corresponding DNAs or vectors orcells expressing same for use in immunotherapy.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein the term “isolated” refers to a compound of interest (forexample a polynucleotide or a polypeptide) that is in an environmentdifferent from that in which the compound naturally occurs e.g.separated from its natural milieu such as by concentrating a peptide toa concentration at which it is not found in nature. “Isolated” includescompounds that are within samples that are substantially enriched forthe compound of interest and/or in which the compound of interest ispartially or substantially purified.

An “immune cell” refers to any cell from the hemopoietic originincluding but not limited to T cells, B cells, monocytes, dendriticcells, and macrophages.

As used herein, the term “polypeptide” refers to a chain of amino acidsof any length, regardless of modification (e.g., phosphorylation orglycosylation).

The term “immune related disease (or disorder or condition)” as usedherein should be understood to encompass any disease disorder orcondition selected from the group including but not limited toautoimmune diseases, inflammatory disorders and immune disordersassociated with graft transplantation rejection, such as acute andchronic rejection of organ transplantation, allogenic stem celltransplantation, autologous stem cell transplantation, bone marrowtranplantation, and graft versus host disease.

As used herein the term “inflammatory disorders” and/or “inflammation”,used interchangeably, includes inflammatory abnormalities characterizedby disregulated immune response to harmful stimuli, such as pathogens,damaged cells, or irritants. Inflammatory disorders underlie a vastvariety of human diseases. Non-immune diseases with etiological originsin inflammatory processes include atherosclerosis, and ischaemic heartdisease. Examples of disorders associated with inflammation include:Chronic prostatitis, Glomerulonephritis, Hypersensitivities, Pelvicinflammatory disease, Reperfusion injury, Sarcoidosis, Vasculitis,Interstitial cystitis, normocomplementemic urticarial vasculitis,pericarditis, myositis, anti-synthetase syndrome, scleritis, macrophageactivation syndrome, Bechet's Syndrome, PAPA Syndrome, Blau's Syndrome,gout, adult and juvenile Still's disease, cryropyrinopathy, Muckle-Wellssyndrome, familial cold-induced auto-inflammatory syndrome, neonatalonset multisystemic inflammatory disease, familial Mediterranean fever,chronic infantile neurologic, cutaneous and articular syndrome, systemicjuvenile idiopathic arthritis, Hyper IgD syndrome, Schnitzler'ssyndrome, TNF receptor-associated periodic syndrome (TRAPSP),gingivitis, periodontitis, hepatitis, cirrhosis, pancreatitis,myocarditis, vasculitis, gastritis, gout, gouty arthritis, andinflammatory skin disorders, selected from the group consisting ofpsoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne.

The term “autoimmune disease” as used herein should be understood toencompass any disease in which recognition of a “self” antigen(self-reactivity) is at least a part of the disease process. Accordingto at least some embodiments of the invention, the autoimmune diseasesshould be understood to encompass any disease disorder or conditionincluding one or more of, but not limited to, multiple sclerosis,rheumatoid arthritis; psoriatic arthritis, discoid lupus erythematosus,systemic lupus erythematosus (SLE); ulcerative colitis; Crohn's disease;benign lymphocytic angiitis, autoimmune lymphoproliferative syndrome,sarcoidosis, autoimmune thrombocytopenic purpura, idiopathicthrombocytopenic purpura, pure red cell aplasia, Sjogren's syndrome,rheumatic disease, polymyalgia rheumatica, mixed connective tissuedisease, inflammatory rheumatism, degenerative rheumatism,extra-articular rheumatism, juvenile arthritis, juvenile rheumatoidarthritis, systemic juvenile idiopathic arthritis, arthritis uratica,muscular rheumatism, chronic polyarthritis, reactive arthritis, Reiter'ssyndrome, rheumatic fever, relapsing polychondritis, Raynaud'sphenomenon, vasculitis, cryoglobulinemic vasculitis, ANCA-associatedvasculitis, temporal arteritis, giant cell arteritis, Takayasuarteritis, Behcet's disease, antiphospholipid syndrome, myastheniagravis, autoimmune haemolytic anaemia, Guillain-Barre syndrome, chronicimmune polyneuropathy, chronic inflammatory demyelinatingpolyneuropathy, autoimmune thyroiditis, insulin dependent diabetesmellitus, type I diabetes, Addison's disease, membranousglomerulonephropathy, polyglandular autoimmune syndromes, Goodpasture'sdisease, autoimmune gastritis, autoimmune atrophic gastritis, perniciousanaemia, pemphigus, pemphigus vulgaris, cirrhosis, primary biliarycirrhosis, idiopathic pulmonary fibrosis, myositis, dermatomyositis,juvenile dermatomyositis, polymyositis, fibromyositis, myogelosis,celiac disease, celiac sprue dermatitis, immunoglobulin A nephropathy,Henoch-Schonlein purpura, Evans syndrome, atopic dermatitis, psoriasis,psoriasis vulgaris, psoriasis arthropathica, Graves' disease, Graves'ophthalmopathy, scleroderma, systemic scleroderma, progressive systemicscleroderma, diffuse scleroderma, localized scleroderma, Crest syndrome,asthma, allergic asthma, allergy, primary biliary cirrhosis, Hashimoto'sthyroiditis, fibromyalgia, chronic fatigue and immune dysfunctionsyndrome (CFIDS), primary myxedema, sympathetic ophthalmia, autoimmuneinner ear disease, autoimmune uveitis, autoimmune chronic activehepatitis, collagen diseases, ankylosing spondylitis, periarthritishumeroscapularis, panarteritis nodosa, polyarteritis nodosa,chondrocalcinosis, Wegener's granulomatosis, microscopic polyangiitis,chronic urticaria, bullous skin disorders, pemphigoid, bullouspemphigoid, cicatricial pemphigoid, vitiligo, atopic eczema, eczema,chronic urticaria, autoimmune urticaria, normocomplementemic urticarialvasculitis, hypocomplementemic urticarial vasculitis, alopecia areata,alopecia universalis, alopecia totalis, Devic's disease, perniciousanemia, childhood autoimmune hemolytic anemia, idiopathic autoimmunehemolytic anemia, refractory or chronic Autoimmune Cytopenias,Prevention of development of Autoimmune Anti-Factor VIII Antibodies inAcquired Hemophilia A, Cold agglutinin disease, Neuromyelitis Optica,Stiff Person Syndrome, gingivitis, periodontitis, pancreatitis,myocarditis, gastritis, gout, gouty arthritis, idiopathic pericarditis,anti-synthetase syndrome, scleritis, macrophage activation syndrome,PAPA Syndrome, Blau's Syndrome, adult and juvenile Still's disease,cryopyrin associated periodic syndrome, Muckle-Wells syndrome, familialcold auto-inflammatory syndrome, neonatal onset multisystem inflammatorydisease, chronic infantile neurologic cutaneous and articular syndrome,familial Mediterranean fever, Hyper IgD syndrome, Schnitzler's syndrome,autoimmune retinopathy, age-related macular degeneration, or TNFreceptor-associated periodic syndrome (TRAPS).

As used herein, “multiple sclerosis” comprises multiple sclerosis or arelated disease, and optionally refers to all types and stages ofmultiple sclerosis, including, but not limited to: benign multiplesclerosis, relapsing remitting multiple sclerosis, secondary progressivemultiple sclerosis, primary progressive multiple sclerosis, progressiverelapsing multiple sclerosis, chronic progressive multiple sclerosis,transitional/progressive multiple sclerosis, rapidly worsening multiplesclerosis, clinically-definite multiple sclerosis, malignant multiplesclerosis, also known as Marburg's Variant, and acute multiplesclerosis. Optionally, “conditions relating to multiple sclerosis”include, e.g., Devic's disease, also known as Neuromyelitis Optica;acute disseminated encephalomyelitis, acute demyelinating opticneuritis, demyelinative transverse myelitis, Miller-Fisher syndrome,encephalomyelradiculoneuropathy, acute demyelinative polyneuropathy,tumefactive multiple sclerosis and Balo's concentric sclerosis.

As used herein, “rheumatoid arthritis” comprises rheumatoid arthritis ora related disease and refers to all types and stages of rheumatoidarthritis, including, but not limited to: rheumatoid arthritis, gout andpseudo-gout, juvenile idiopathic arthritis, juvenile rheumatoidarthritis, Still's disease, ankylosing spondylitis, rheumatoidvasculitis. Optionally, conditions relating to rheumatoid arthritisinclude, e.g., osteoarthritis, sarcoidosis, Henoch-Schönlein purpura,Psoriatic arthritis, Reactive arthritis, Spondyloarthropathy, septicarthritis, Haemochromatosis, Hepatitis, vasculitis, Wegener'sgranulomatosis, Lyme disease, Familial Mediterranean fever,Hyperimmunoglobulinemia D with recurrent fever, TNF receptor associatedperiodic syndrome, and Enteropathic arthritis associated withinflammatory bowel disease.

As used herein, “Uveitis” refers to all types and stages of Uveitis,including, but not limited to: anterior uveitis (or iridocyclitis),intermediate uveitis (pars planitis), posterior uveitis (orchorioretinitis) and the panuveitic form.

As used herein, “inflammatory bowel disease” also refers to a relateddisease and refers to all types and stages of inflammatory bowel disease(IBD), including, but not limited to: Crohn's disease and ulcerativecolitis (UC). Optionally, conditions relating to IBD include, e.g.,Collagenous colitis, Lymphocytic colitis, Ischaemic colitis, Diversioncolitis, Behçet's disease, Indeterminate colitis.

As used herein, “psoriasis” also refers to a related disease and refersto all types and stages of psoriasis, including, but not limited to:Nonpustular Psoriasis including Psoriasis vulgaris and Psoriaticerythroderma (erythrodermic psoriasis), Pustular psoriasis includingGeneralized pustular psoriasis (pustular psoriasis of von Zumbusch),Pustulosis palmaris et plantaris (persistent palmoplantar pustulosis,pustular psoriasis of the Barber type, pustular psoriasis of theextremities), Annular pustular psoriasis, Acrodermatitis continua,Impetigo herpetiformis. Optionally, conditions relating to psoriasisinclude, e.g., drug-induced psoriasis, Inverse psoriasis, Napkinpsoriasis, Seborrheic-like psoriasis, Guttate psoriasis, Nail psoriasis,Psoriatic arthritis.

As used herein, “type 1 diabetes” refers to all types and stages of type1 diabetes, including, but not limited to: insulin-dependent diabetesmellitus, idiopathic diabetes, juvenile type ldiabetes, maturity onsetdiabetes of the young, latent autoimmune diabetes in adults, gestationaldiabetes. Conditions relating to type 1 diabetes include, neuropathyincluding polyneuropathy, mononeuropathy, peripheral neuropathy andautonomicneuropathy; eye complications: glaucoma, cataracts,retinopathy.

As used herein, “Sjogren's syndrome” refers to all types and stages ofSjogren's syndrome, including, but not limited to: Primary Sjogren'ssyndrome and Secondary Sjogren's syndrome. Conditions relating toSjogren's syndrome include connective tissue disease, such as rheumatoidarthritis, systemic lupus erythematosus, or scleroderma. Othercomplications include pneumonia, polmunary fibrosis, interstitialnephritis, inflammation of the tissue around the kidney's filters,glomerulonephritis, renal tubular acidosis, carpal tunnel syndrome,peripheral neuropathy, cranial neuropathy, primary biliary cirrhosis(PBC), cirrhosis, Inflammation in the esophagus, stomach, pancreas, andliver (including hepatitis), Polymyositis, Raynaud's phenomenon,Vasculitis, Autoimmune thyroid problems, lymphoma.

As used herein, “systemic lupus erythematosus”, refers to all types andstages of systemic lupus erythematosus, including, but not limited todiscoid lupus, lupus arthritis, lupus pneumonitis, lupus nephritis.Conditions relating to systemic lupus erythematosus includeosteoarticular tuberculosis, antiphospholipid antibody syndrome,inflammation of various parts of the heart, such as pericarditis,myocarditis, and endocarditis, Lung and pleura inflammation, pleuritis,pleural effusion, chronic diffuse interstitial lung disease, pulmonaryhypertension, pulmonary emboli, pulmonary hemorrhage, and shrinking lungsyndrome, lupus headache, Guillain-Barré syndrome, aseptic meningitis,demyelinating syndrome, mononeuropathy, mononeuritis multiplex,myasthenia gravis, myelopathy, cranial neuropathy, polyneuropathy,vasculitis.

As used herein the term “infectious disorder and/or disease” and/or“infection”, used interchangeably, includes any disorder, disease and/orcondition caused by presence and/or growth of pathogenic biologicalagent in an individual host organism. As used herein the term“infection” comprises the disorder, disease and/or condition as above,exhibiting clinically evident illness (i.e., characteristic medicalsigns and/or symptoms of disease) and/or which is asymtomatic for muchor all of it course. As used herein the term “infection” also comprisesdisorder, disease and/or condition caused by persistence of foreignantigen that lead to exhaustion T cell phenotype characterized byimpaired functionality which is manifested as reduced proliferation andcytokine production. As used herein the term “infectious disorder and/ordisease” and/or “infection”, further includes any of the below listedinfectious disorders, diseases and/or conditions, caused by a bacterialinfection, viral infection, fungal infection and/or parasite infection.

As used herein the term “viral infection” comprises any infection causedby a virus, optionally including but not limited to Retroviridae (e.g.,human immunodeficiency viruses, such as HIV-1 or HIV-2, acquired immunedeficiency (AIDS) also referred to as HTLV-III, LAV or HTLV-III/LAV, orHIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polioviruses, hepatitis A virus; enteroviruses, human coxsackie viruses,rhinoviruses, echoviruses); Calciviridae (e.g., strains that causegastroenteritis); Togaviridae (e.g., equine encephalitis viruses,rubella viruses); Flaviridae (e.g., dengue viruses, encephalitisviruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses);Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses);Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenzaviruses, mumps virus, measles virus, respiratory syncytial virus);Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaanviruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae(hemorrhagic fever virus); Reoviridae (e.g., reoviruses, orbiviruses androtaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyomaviruses); Adenoviridae (most adenoviruses); Herperviridae (herpessimplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus(CMV), herpes viruses); Poxviridae (variola virsues, vaccinia viruses,pox viruses); and Iridoviridae (e.g., African swine fever virus); andunclassified viruses (e.g., the etiological agents of Spongiformencephalopathies, the agent of delta hepatitides (thought to be adefective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1—internally transmitted; class 2—parenterallytransmitted (i.e., Hepatitis C); Norwalk and related viruses, andastroviruses) as well as Severe acute respiratory syndrome virus andrespiratory syncytial virus (RSV).

As used herein the term “fungal infection” comprises any infectioncaused by a fungi, optionally including but not limited to Cryptococcusneoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomycesdermatitidis, Chlamydia trachomatis, Candida albicans.

As used herein the term “parasite infection” comprises any infectioncaused by a parasite, optionally including but not limited to protozoa,such as Amebae, Flagellates, Plasmodium falciparum, Toxoplasma gondii,Ciliates, Coccidia, Microsporidia, Sporozoa; helminthes, Nematodes(Roundworms), Cestodes (Tapeworms), Trematodes (Flukes), Arthropods, andaberrant proteins known as prions.

An infectious disorder and/or disease caused by bacteria may optionallycomprise one or more of Sepsis, septic shock, sinusitis, skininfections, pneumonia, bronchitis, meningitis, Bacterial vaginosis,Urinary tract infection (UCI), Bacterial gastroenteritis, Impetigo anderysipelas, Erysipelas, Cellulitis, anthrax, whooping cough, lymedisease, Brucellosis, enteritis, acute enteritis, Tetanus, diphtheria,Pseudomembranous colitis, Gas gangrene, Acute food poisoning, Anaerobiccellulitis, Nosocomial infections, Diarrhea, Meningitis in infants,Traveller's diarrhea, Hemorrhagic colitis, Hemolytic-uremic syndrome,Tularemia, Peptic ulcer, Gastric and Duodenal ulcers, Legionnaire'sDisease, Pontiac fever, Leptospirosis, Listeriosis, Leprosy (Hansen'sdisease), Tuberculosis, Gonorrhea, Ophthalmia neonatorum, Septicarthritis, Meningococcal disease including meningitis,Waterhouse-Friderichsen syndrome, Pseudomonas infection, Rocky mountainspotted fever, Typhoid fever type salmonellosis, Salmonellosis withgastroenteritis and enterocolitis, Bacillary dysentery/Shigellosis,Coagulase-positive staphylococcal infections: Localized skin infectionsincluding Diffuse skin infection (Impetigo), Deep localized infections,Acute infective endocarditis, Septicemia, Necrotizing pneumonia,Toxinoses such as Toxic shock syndrome and Staphylococcal foodpoisoning, Cystitis, Endometritis, Otitis media, Streptococcalpharyngitis, Scarlet fever, Rheumatic fever, Puerperal fever,Necrotizing fasciitis, Cholera, Plague (including Bubonic plague andPneumonic plague), as well as any infection caused by a bacteriaselected from but not limited to Helicobacter pyloris, Boreliaiburgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M.tuberculosis, M. avium, M. Intracellulare, M. kansaii, M gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp Haemophilus influenzae, Bacillusantracis, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter erogenes, Klebsiella pneuomiae, Pasteurellamulticoda, Bacteroides sp., Fusobacterium nucleatum, Sreptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira, andActinomeyces israelli.

Non limiting examples of infectious disorder and/or disease caused byvirus is selected from the group consisting of but not limited toacquired immune deficiency (AIDS), West Nile encephalitis, coronavirusinfection, rhinovirus infection, influenza, dengue, hemorrhagic fever;an otological infection; severe acute respiratory syndrome (SARS), acutefebrile pharyngitis, pharyngoconjunctival fever, epidemickeratoconjunctivitis, infantile gastroenteritis, infectiousmononucleosis, Burkitt lymphoma, acute hepatitis, chronic hepatitis,hepatic cirrhosis, hepatocellular carcinoma, primary HSV-1 infection,(gingivostomatitis in children, tonsillitis & pharyngitis in adults,keratoconjunctivitis), latent HSV-1 infection (herpes labialis, coldsores), aseptic meningitis, Cytomegalovirus infection, Cytomegalicinclusion disease, Kaposi sarcoma, Castleman disease, primary effusionlymphoma, influenza, measles, encephalitis, postinfectiousencephalomyelitis, Mumps, hyperplastic epithelial lesions (common, flat,plantar and anogenital warts, laryngeal papillomas, epidermodysplasiaverruciformis), croup, pneumonia, bronchiolitis, Poliomyelitis, Rabies,bronchiolitis, pneumonia, German measles, congenital rubella,Hemorrhagic Fever, Chickenpox, Dengue, Ebola infection, Echovirusinfection, EBV infection, Fifth Disease, Filovirus, Flavivirus, Hand,foot & mouth disease, Herpes Zoster Virus (Shingles), Human PapillomaVirus Associated Epidermal Lesions, Lassa Fever, Lymphocyticchoriomeningitis, Parainfluenza Virus Infection, Paramyxovirus,Parvovirus B19 Infection, Picornavirus, Poxviruses infection, Rotavirusdiarrhea, Rubella, Rubeola, Varicella, Variola infection.

An infectious disorder and/or disease caused by fungi optionallyincludes but is not limited to Allergic bronchopulmonary aspergillosis,Aspergilloma, Aspergillosis, Basidiobolomycosis, Blastomycosis,Candidiasis, Chronic pulmonary aspergillosis, Chytridiomycosis,Coccidioidomycosis, Conidiobolomycosis, Covered smut (barley),Cryptococcosis, Dermatophyte, Dermatophytid, Dermatophytosis, Endothrix,Entomopathogenic fungus, Epizootic lymphangitis, Epizootic ulcerativesyndrome, Esophageal candidiasis, Exothrix, Fungemia, Histoplasmosis,Lobomycosis, Massospora cicadina, Mycosis, Mycosphaerella fragariae,Myringomycosis, Paracoccidioidomycosis, Pathogenic fungi, Penicilliosis,Thousand cankers disease, Tinea, Zeaspora, Zygomycosis.Non limitingexamples of infectious disorder and/or disease caused by parasites isselected from the group consisting of but not limited to Acanthamoeba,Amoebiasis, Ascariasis, Ancylostomiasis, Anisakiasis, Babesiosis,Balantidiasis, Baylisascariasis, Blastocystosis, Candiru, Chagasdisease, Clonorchiasis, Cochliomyia, Coccidia, Chinese Liver FlukeCryptosporidiosis, Dientamoebiasis, Diphyllobothriasis, Dioctophymerenalis infection, Dracunculiasis, Echinococcosis, Elephantiasis,Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis,Gnathostomiasis, Hymenolepiasis, Halzoun Syndrome, Isosporiasis,Katayama fever, Leishmaniasis, lymphatic filariasis, Malaria,Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Primary amoebicmeningoencephalitis, Parasitic pneumonia, Paragonimiasis, Scabies,Schistosomiasis, Sleeping sickness, Strongyloidiasis, Sparganosis,Rhinosporidiosis, River blindness, Taeniasis (cause of Cysticercosis),Toxocariasis, Toxoplasmosis, Trichinosis, Trichomoniasis, Trichuriasis,Trypanosomiasis, Tapeworm infection.

A preferred example of infectious disease is a disease caused by any ofhepatitis B, hepatitis C, infectious mononucleosis, EBV,cytomegalovirus, AIDS, HIV-1, HIV-2, tuberculosis, malaria andschistosomiasis.

As used herein, the term “vaccine” refers to a biological preparationthat improves immunity to a particular disease, wherein the vaccineincludes an antigen, such as weakened or killed forms of pathogen, itstoxins or one of its surface proteins, against which immune responsesare elicited. A vaccine typically includes an adjuvant as immunepotentiator to stimulate the immune system. As used herein, the term“therapeutic vaccine” and/or “therapeutic vaccination” refers to avaccine used to treat ongoing disease, such as infectious disease.

As used herein, the term “adjuvant” refers to an agent used to stimulatethe immune system and increase the response to a vaccine, without havingany specific antigenic effect in itself.

As used herein, a “costimulatory polypeptide” or “costimulatorymolecule” is a polypeptide that, upon interaction with a cell-surfacemolecule on T cells, modulates T cell responses.

As used herein, a “costimulatory signaling” is the signaling activityresulting from the interaction between costimulatory polypeptides onantigen presenting cells and their receptors on T cells duringantigen-specific T cell responses. Without wishing to be limited by asingle hypothesis, the antigen-specific T cell response is believed tobe mediated by two signals: 1) engagement of the T cell Receptor (TCR)with antigenic peptide presented in the context of MHC (signal 1), and2) a second antigen-independent signal delivered by contact betweendifferent costimulatory receptor/ligand pairs (signal 2). Withoutwishing to be limited by a single hypothesis, this “second signal” iscritical in determining the type of T cell response (activation vsinhibition) as well as the strength and duration of that response, andis regulated by both positive and negative signals from costimulatorymolecules, such as the B7 family of proteins.

As used herein, the term “B7” polypeptide means a member of the B7family of proteins that costimulate T cells including, but not limitedto B7-1, B7-2, B7-DC, B7-H5, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-S3and biologically active fragments and/or variants thereof.Representative biologically active fragments include the extracellulardomain or fragments of the extracellular domain that costimulate Tcells.

As used herein, “inflammatory molecules” refers to molecules that induceinflammatory responses (directly or indirectly) including, but notlimited to, cytokines and metalloproteases such as including, but notlimited to, IL-1beta, TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6,IL-23, IL-22, IL-21, and MMPs.

As used herein, the term “vaccine” refers to a biological preparationthat improves immunity to a particular disease, wherein the vaccineincludes an antigen, such as weakened or killed forms of pathogen, itstoxins or one of its surface proteins, against which immune responsesare elicited. A vaccine typically includes an adjuvant as immunepotentiator to stimulate the immune system. As used herein, the term“therapeutic vaccine” and/or “therapeutic vaccination” refers to avaccine used to treat ongoing disease, such as infectious disease.

As used herein, the term “adjuvant” refers to an agent used to stimulatethe immune system and increase the response to a vaccine, without havingany specific antigenic effect in itself.

As used herein, a “vector” is a replicon, such as a plasmid, phage, orcosmid, into which another DNA segment may be inserted so as to bringabout the replication of the inserted segment. The vectors describedherein can be expression vectors. As used herein, an “expression vector”is a vector that includes one or more expression control sequences

As used herein, an “expression control sequence” is a DNA sequence thatcontrols and regulates the transcription and/or translation of anotherDNA sequence.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usual orintended function. Thus, two different polypeptides operably linkedtogether retain their respective biological functions while physicallylinked together.

As used herein, “valency” refers to the number of binding sitesavailable per molecule.

As used herein, a “variant” polypeptide contains at least one amino acidsequence alteration as compared to the amino acid sequence of thecorresponding wild-type polypeptide.

As used herein, “conservative” amino acid substitutions aresubstitutions wherein the substituted amino acid has similar structuralor chemical properties. As used herein, the term “host cell” refers toprokaryotic and eukaryotic cells into which a recombinant vector can beintroduced.

As used herein, “transformed” and “transfected” encompass theintroduction of a nucleic acid (e.g. a vector) into a cell by a numberof techniques known in the art.

As used herein, the terms “immunologic”, “immunological” or “immune”response is the development of a beneficial humoral (antibody mediated)and/or a cellular (mediated by antigen-specific T cells or theirsecretion products) response directed against a peptide in a recipientpatient. Such a response can be an active response induced byadministration of immunogen or a passive response induced byadministration of antibody or primed T-cells. Without wishing to belimited by a single hypothesis, a cellular immune response is elicitedby the presentation of polypeptide epitopes in association with Class Ior Class II MHC molecules to activate antigen-specific CD4+ T helpercells and/or CD8+ cytotoxic T cells. The response may also involveactivation of monocytes, macrophages, NK cells, basophils, dendriticcells, astrocytes, microglia cells, eosinophils, activation orrecruitment of neutrophils or other components of innate immunity. Thepresence of a cell-mediated immunological response can be determined byproliferation assays (CD4+ T cells) or CTL (cytotoxic T lymphocyte)assays. The relative contributions of humoral and cellular responses tothe protective or therapeutic effect of an immunogen can bedistinguished by separately isolating antibodies and T-cells from animmunized syngeneic animal and measuring protective or therapeuticeffect in a second subject.

An “immunogenic agent” or “immunogen” is capable of inducing animmunological response against itself on administration to a mammal,optionally in conjunction with an adjuvant.

As used herein, the term “C1ORF32” refers to the protein encoded by anyone of the H19011_1_T8 (SEQ ID NO:1), H19011_1_T9 (SEQ ID NO:2)transcripts reported herein, particularly to proteins as set forth inany one of H19011_1_P8 (SEQ ID NO:4), H19011_1_P8_V1 (SEQ ID NO:5),H19011_1_P9 (SEQ ID NO:6) or H19011_1_P9_V1 (SEQ ID NO:34), variants andfragments thereof, which can have therapeutic effect on immune relateddisorder and/or infection.

Fragments of C1ORF32 Polypeptides

As used herein the term “soluble C1ORF32” or “soluble C1ORF32proteins/molecules” refers to fragments of C1ORF32 that include some orall of the IgV domain of the C1ORF32 polypeptide, and lack some or allof the intracellular and/or transmembrane domains, wherein saidfragments retain a biological activity of inhibition of T cellactivation.

The soluble C1ORF32 molecules used in the methods of the invention mayor may not include a signal (leader) peptide sequence.

Various fragments are given in Table 1 below. “N-term” refers to theN-terminus, so “first N-term” refers to the N-terminus. Particularsequences of interest, according to at least some embodiments of thepresent invention, include but are not limited to SEQ ID NOs: 29, 30,41-105, and/or 45, 64, or 96. It should be noted that one or moreactivities and/or functions ascribed herein to any of SEQ ID NOs: 29,30, 41-105 are also applicable to any of SEQ ID NOs: 45, 64 or 96.

The below table describes a number of sequences with abbreviations,which are defined as follows. The terms “first N-term” and “secondN-term” refer to the two potential start points of the IgV domain, asshown in FIG. 1. The terms “#1 option cut point” (and “#2 option cutpoint”) refer to different possible C-terminal end points of the solubleprotein—also as shown in FIG. 1. FIG. 1 shows five different possibleC-terminal end points, which are as follows: the end of the IgV domain,#1 option cut point, #2 option cut point, the end of the producedprotein, and the position before the start of the predicted TM(transmembrane region, which occurs two amino acids after the end of theproduced protein). Reference to “+1” or “−1” from a reference pointindicate one amino acid after or before that point, respectively, withlarger numbers indicating a greater number of amino acids after orbefore that point as stated below.

The right-most column relates to the SEQ ID NO of the amino acidsequence fused to an Fc sequence (hIgG1C220S-SEQ ID NO 115) to form afusion protein; for example, the amino acid sequence having SEQ IDNO:14, given as fused the above Fc sequence, has SEQ ID NO:116 as afusion protein.

TABLE 1 amino acid sequences discussed in the present applicationSEQ ID NO with Fc SEQ (hIgG1 C220S - ID SEQ ID 115) NO:Amino acid sequence Description fusion 14LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV residues 21-184 of 116VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE H19011_1_P8WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG REITIVHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNEGSLGLLVLGRTGLLADLLPSFAVEIM 15 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVresidues 21-169 of 117 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE H19011_1_P9WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG REITIVHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNEGSLGLLVLEWV 19 MDRVLLRWISLFWLTAMVEGLQVTVPDKKKVAMresidues 1-184 of the 118 LFQPTVLRCHFSTSSHQPAVVQWKFKSYCQDRMsequence H19011_1_P8_V1 GESLGMSSTRAQSLSKRNLEWDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRGREITIVHDADLQI GKLMWGDSGLYYCIITTPDDLEGKNEDSVELLVLGRTGLLADLLPSFAVEIM 28 MDRVLLRWISLFWLTAMVEGLQVTVPDKKKVAMresidues 1-169 of 119 LFQPTVLRCHFSTSSHQPAVVQWKFKSYCQDRM H19011_1_P9GESLGMSSTRAQSLSKRNLEWDPYLDCLDSRRT VRVVASKQGSTVTLGDFYRGREITIVHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNEGSLGLLV LEW 29LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV residues 21-167 of 120VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE H19011_1_P8_V1WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG REITIVHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNEDSVELLVLG 30 CHFSTSSHQPAVVQWKFKSYCQDRMGESLGMSS residues 42-145 of121 TRAQSLSKRNLEWDPYLDCLDSRRTVRVVASKQ (H19011_1_P8 fromGSTVTLGDFYRGREITIVHDADLQIGKLMWGDS first C residue to GLYYClast C residue) 35 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVH19011_1_P8_V1_from_21_ 122 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLEto_184 (first N- WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to end ofREITIVHDADLQIGKLMWGDSGLYYCIITTPDD produced ECD −2 fromLEGKNEDSVELLVLGRTGLLADLLPSFAVEIM end of predicted ECD) 36LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV residues 21-169 of 123VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE H19011_1_P9_V1WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG REITIVHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNEDSVELLVLEWV 37 MDRVLLRWISLFWLTAMVEGLQVTVPDKKKVAM residues 1-184 of124 LFQPTVLRCHFSTSSHQPAVVQWKFKSYCQDRM H19011_1_P8GESLGMSSTRAQSLSKRNLEWDPYLDCLDSRRT VRVVASKQGSTVTLGDFYRGREITIVHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNEGSLGLLV LGRTGLLADLLPSFAVEIM 40MDRVLLRWISLFWLTAMVEGLQVTVPDKKKVAM residues 1-169 of 125LFQPTVLRCHFSTSSHQPAVVQWKFKSYCQDRM H19011_1_P9_V1GESLGMSSTRAQSLSKRNLEWDPYLDCLDSRRT VRVVASKQGSTVTLGDFYRGREITIVHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNEDSVELLV LEWV 41LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV residues 21-167 of 126VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE H19011_1_P8 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to end of IgV)REITIVHDADLQIGKLMWGDSGLYYCIITTPDD LEGKNEGSLGLLVLG 42LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 127VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE l_to_164 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to −3 from endREITIVHDADLQIGKLMWGDSGLYYCIITTPDD of IgV) LEGKNEDSVELL 43LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 128VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE l_to_165 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to −2 from endREITIVHDADLQIGKLMWGDSGLYYCIITTPDD of IgV) LEGKNEDSVELLV 44LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 129VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_166 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to −1 from endREITIVHDADLQIGKLMWGDSGLYYCIITTPDD of IgV) LEGKNEDSVELLVL 45LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 130VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_184 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to end ofREITIVHDADLQIGKLMWGDSGLYYCIITTPDD produced ECD with FA- >LEGKNEDSVELLVLGRTGLLADLLPSGGVEIM GG mutation) 46LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 131VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_168 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to +1 after endREITIVHDADLQIGKLMWGDSGLYYCIITTPDD of IgV) LEGKNEDSVELLVLGR 47LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 132VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_169 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to +2 after endREITIVHDADLQIGKLMWGDSGLYYCIITTPDD of IgV) LEGKNEDSVELLVLGRT 48LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 133VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_170 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to +3 after endREITIVHDADLQIGKLMWGDSGLYYCIITTPDD of IgV −3 before #1 LEGKNEDSVELLVLGRTGoption cut point) 49 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVH19011_1_P8_V1_from_21_ 134 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLEto_171 (first N- WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to −2 before #1REITIVHDADLQIGKLMWGDSGLYYCIITTPDD option cut point) LEGKNEDSVELLVLGRTGL50 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 135VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_172 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to −1 before #1REITIVHDADLQIGKLMWGDSGLYYCIITTPDD option cut point) LEGKNEDSVELLVLGRTGLL51 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 136VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_173 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to #1 option cutREITIVHDADLQIGKLMWGDSGLYYCIITTPDD point) LEGKNEDSVELLVLGRTGLLA 52LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 137VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_174 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to +1 after #1REITIVHDADLQIGKLMWGDSGLYYCIITTPDD option cut point)LEGKNEDSVELLVLGRTGLLAD 53 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVH19011_1_P8_V1_from_21_ 138 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLEto_175 (first N- WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to +2 after #1REITIVHDADLQIGKLMWGDSGLYYCIITTPDD option cut point −3LEGKNEDSVELLVLGRTGLLADL before #2 option cut point) 54LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 139VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_176 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to +3 after #1REITIVHDADLQIGKLMWGDSGLYYCIITTPDD option cut point −2LEGKNEDSVELLVLGRTGLLADLL before #2 option cut point) 55LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 140VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_177 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to −1 before #2REITIVHDADLQIGKLMWGDSGLYYCIITTPDD option cut point)LEGKNEDSVELLVLGRTGLLADLLP 56 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVH19011_1_P8_V1_from_21_ 141 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLEto_178 (first N- WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to #2 option cutREITIVHDADLQIGKLMWGDSGLYYCIITTPDD point LEGKNEDSVELLVLGRTGLLADLLPS 57LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 142VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_179 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to +1 after #2REITIVHDADLQIGKLMWGDSGLYYCIITTPDD option cut point)LEGKNEDSVELLVLGRTGLLADLLPSF 58 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVH19011_1_P8_V1_from_21_ 143 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLEto_180 (first N- WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to +2 after #2REITIVHDADLQIGKLMWGDSGLYYCIITTPDD option cut point)LEGKNEDSVELLVLGRTGLLADLLPSFA 59 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVH19011_1_P8_V1_from_21_ 144 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLEto_181 (first N- WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to +3 after #2REITIVHDADLQIGKLMWGDSGLYYCIITTPDD option cut point −3LEGKNEDSVELLVLGRTGLLADLLPSFAV from end of produced ECD) 60LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 145VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_182(first N-termWDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG to −2 from end ofREITIVHDADLQIGKLMWGDSGLYYCIITTPDD produced ECD)LEGKNEDSVELLVLGRTGLLADLLPSFAVE 61 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVH19011_1_P8_V1_from_21_ 146 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLEto_183 (first N- WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to −1 from endREITIVHDADLQIGKLMWGDSGLYYCIITTPDD of produced ECD −3LEGKNEDSVELLVLGRTGLLADLLPSFAVEI from end of predicted ECD) 62LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1 from 147VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE 21 to 186 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to end ofREITIVHDADLQIGKLMWGDSGLYYCIITTPDD predicted ECD)LEGKNEDSVELLVLGRTGLLADLLPSFAVEIMP E 63 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVH19011_1_P8_V1_from_21_ 148 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLEto_185 (first N- WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to +1 afterREITIVHDADLQIGKLMWGDSGLYYCIITTPDD produced ECD −1 fromLEGKNEDSVELLVLGRTGLLADLLPSFAVEIMP end of predicted ECD) 64LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 149VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_184 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to end ofREITIVHDADLQIGKLMWGDSGLYYCIITTPDD produced ECD with FA- >LEGKNEDSVELLVLGRTGLLADLLPSGAVEIM GA mutation) 65LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 150VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_187(first N-termWDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG to +3 after producedREITIVHDADLQIGKLMWGDSGLYYCIITTPDD ECD +1 in predictedLEGKNEDSVELLVLGRTGLLADLLPSFAVEIMP TM) EW 66LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 151VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_188 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to +2 inREITIVHDADLQIGKLMWGDSGLYYCIITTPDD predicted TM)LEGKNEDSVELLVLGRTGLLADLLPSFAVEIMP EWV 67LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 152VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_189 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to +3 inREITIVHDADLQIGKLMWGDSGLYYCIITTPDD predicted TM)LEGKNEDSVELLVLGRTGLLADLLPSFAVEIMP EWVF 68DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 153SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_164(second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to −3 from endHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE of IgV) DSVELL 69DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 154SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_165(second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to −2 from endHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE of IgV) DSVELLV 70DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 155SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_166 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to −1 from endHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE of IgV) DSVELLVL 71DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 156SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_167 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to end of IgV)HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE DSVELLVLG 72DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 157SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_168 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +1 after endHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE of IgV) DSVELLVLGR 73DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 158SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_169 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +2 after endHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE of IgV) DSVELLVLGRT 74DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 159SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_170 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +3 after endHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE of IgV −3 before #1 DSVELLVLGRTGoption cut point) 75 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFKH19011_1_P8_V1_from_27_ 160 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLDto_171 (second N- CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to −2 before #1HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE option cut point) DSVELLVLGRTGL 76DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 161SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_172 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to −1 before #1HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE option cut point) DSVELLVLGRTGLL 77DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 162SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_173 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to #1 option cutHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE point) DSVELLVLGRTGLLA 78DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 163SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_174 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +1 after #1HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE option cut point) DSVELLVLGRTGLLAD 79DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 164SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_175 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +2 after #1HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE option cut point −3 DSVELLVLGRTGLLADLbefore #2 option cut point) 80 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFKH19011_1_P8_V1_from_27_ 165 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLDto_176 (second N- CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +3 after #1HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE option cut point −2 DSVELLVLGRTGLLADLLbefore #2 option cut point) 81 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFKH19011_1_P8_V1_from_27_ 166 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLDto_177 (second N- CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to −1 before #2HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE option cut point) DSVELLVLGRTGLLADLLP82 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 167SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_178 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to #2 option cutHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE point) DSVELLVLGRTGLLADLLPS 83DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 168SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_179 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +1 after #2HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE option cut point)DSVELLVLGRTGLLADLLPSF 84 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFKH19011_1_P8_V1_from_27_ 169 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLDto_180 (second N- CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +2 after #2HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE option cut point)DSVELLVLGRTGLLADLLPSFA 85 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFKH19011_1_P8_V1_from_27_ 170 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLDto_181 (second N- CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +3 after #2HDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE option cut point −3DSVELLVLGRTGLLADLLPSFAV from end of produced ECD) 86DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 171SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_182 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to −2 from endHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE of produced ECD)DSVELLVLGRTGLLADLLPSFAVE 87 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFKH19011_1_P8_V1_from_27_ 172 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLDto_183 (second N- CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to −1 from endHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE of produced ECD −3DSVELLVLGRTGLLADLLPSFAVEI from end of predicted ECD) 88DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 173SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_184 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to end ofHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE produced ECD −2 fromDSVELLVLGRTGLLADLLPSFAVEIM end of predicted ECD) 89DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 174SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_185 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +1 after endHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE of produced ECD −1DSVELLVLGRTGLLADLLPSFAVEIMP from end of predicted ECD) 90DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 175SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_186 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +2 after endHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE of produced ECD endDSVELLVLGRTGLLADLLPSFAVEIMPE of predicted ECD) 91DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 176SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_187 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +2 after endHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE of produced ECD +1 inDSVELLVLGRTGLLADLLPSFAVEIMPEW predicted TM) 92DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P8_V1_from_27_ 177SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_188 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +2 inHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE predicted TM)DSVELLVLGRTGLLADLLPSFAVEIMPEWV 93 DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFKH19011_1_P8_V1_from_27_ 178 SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLDto_189 (second N- CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +3 inHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE predicted TM)DSVELLVLGRTGLLADLLPSFAVEIMPEWVF 94 LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAVH19011_1_P9_V1_from_21_ 179 VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLEto_167 (first N- WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to −2 from endREITIVHDADLQIGKLMWGDSGLYYCIITTPDD of predicted ECD) LEGKNEDSVELLVLE 95LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P9_V1_from_21_ 180VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_168 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to −1 from endREITIVHDADLQIGKLMWGDSGLYYCIITTPDD of predicted ECD) LEGKNEDSVELLVLEW 96LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P8_V1_from_21_ 181VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_184 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to end ofREITIVHDADLQIGKLMWGDSGLYYCIITTPDD produced ECD with FA- >LEGKNEDSVELLVLGRTGLLADLLPSAAVEIM AA mutation) 97LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P9_V1_from_21_ 182VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_170 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to +1 predictedREITIVHDADLQIGKLMWGDSGLYYCIITTPDD TM) LEGKNEDSVELLVLEWVF 98LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P9_V1_from_21_ 183VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_171 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to +2 predictedREITIVHDADLQIGKLMWGDSGLYYCIITTPDD TM) LEGKNEDSVELLVLEWVFV 99LQVTVPDKKKVAMLFQPTVLRCHFSTSSHQPAV H19011_1_P9_V1_from_21_ 184VQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLE to_172 (first N-WDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRG term to +3 predictedREITIVHDADLQIGKLMWGDSGLYYCIITTPDD TM) LEGKNEDSVELLVLEWVFVG 100DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P9_V1_from_27_ 185SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_167 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to −2 from endHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE of predicted ECD) DSVELLVLE 101DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P9_V1_from_27_ 186SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_168 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to −1 from endHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE of predicted ECD) DSVELLVLEW 102DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P9_V1_from_27_ 187SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_169 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to end ofHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE predicted ECD) DSVELLVLEWV 103DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P9_V1_from_27_ 188SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_170 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +1 predictedHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE TM) DSVELLVLEWVF 104DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P9_V1_from_27_ 189SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_171 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +2 predictedHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE TM) DSVELLVLEWVFV 105DKKKVAMLFQPTVLRCHFSTSSHQPAVVQWKFK H19011_1_P9_V1_from_27_ 190SYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLD to_172 (second N-CLDSRRTVRVVASKQGSTVTLGDFYRGREITIV term to +3 predictedHDADLQIGKLMWGDSGLYYCIITTPDDLEGKNE TM) DSVELLVLEWVFVG

In particular, the fragments of the extracellular domain of C1ORF32 caninclude any sequence corresponding to any portion of or comprising theIgV domain of the extracellular domain of C1ORF32, having any sequencecorresponding to residues of H19011_1_P8 (SEQ ID NO:4) starting from anyposition between 18 and 31 and ending at any position between 157 and175 or corresponding to residues of H19011_1_P8_V1 (SEQ ID NO:5)starting from any position between 18 and 31 and ending at any positionbetween 157 and 175, or corresponding to residues of H19011_1_P9 (SEQ IDNO:6) starting from any position between 18 and 31 and ending at anyposition between 159 and 172, or corresponding to residues ofH19011_1_P9_V1 (SEQ ID NO:34) starting from any position between 18 and31 and ending at any position between 159 and 172. The base sequences asgiven above are without a signal peptide.

The C1ORF32 proteins contain an immunoglobulin domain within theextracellular domain, the IgV domain (or V domain), which is related tothe variable domain of antibodies. The IgV domain may be responsible forreceptor binding, by analogy to the other B7 family members. The Igdomain of the extracellular domain includes one disulfide bond formedbetween intradomain cystein residues, as is typical for this fold andmay be important for structure-function. In SEQ ID NO: 4 these cysteinesare located at residues 42 and 145.

In one embodiment, the first fusion partner is a soluble fragment ofC1ORF32. Without wishing to be limited by a single hypothesis, it isbelieved that useful fragments are those that retain the ability to bindto their natural receptor or receptors and/or retain the ability toinhibit T cell activation. A C1ORF32 polypeptide that is a fragment offull-length C1ORF32 typically has at least 20 percent, 30 percent, 40percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95percent, 98 percent, 99 percent, 100 percent, or even more than 100percent of the ability to bind its natural receptor(s) and/or of theability to inhibit T cell activation as compared to full-length C1ORF32.Soluble C1ORF32 polypeptide fragments are fragments of C1ORF32polypeptides that may be shed, secreted or otherwise extracted from theproducing cells. In other embodiments, the soluble fragments of C1ORF32polypeptides include fragments of the C1ORF32 extracellular domain thatretain C1ORF32 biological activity, such as fragments that retain theability to bind to their natural receptor or receptors and/or retain theability to inhibit T cell activation. The extracellular domain caninclude 1, 2, 3, 4, or 5 contiguous amino acids from the transmembranedomain, and/or 1, 2, 3, 4, or 5 contiguous amino acids from the signalsequence. Alternatively, the extracellular domain can have 1, 2, 3, 4, 5or more amino acids removed from the C-terminus, N-terminus, or both.

In some embodiments the extracellular domain is only the IgV domain asset forth in SEQ ID NO: 29, or fragments or variants thereof, or theregion between the conserved cysteines of the IgV domain which arelocated at residues 42 and 145 of the full-length protein SEQ ID NO:4,corresponding to the sequence set forth in SEQ ID NO: 30:CHFSTSSHQPAVVQWKFKSYCQDRMGESLGMSSTRAQSLSKRNLEWDPYLDCLDSRRTVRVVASKQGSTVTLGDFYRGREITIVHDADLQIGKLMWGDSGLYYC. In particular, thefragments of the IgV domain can include any sequence corresponding toresidues of H19011_1_P8 (SEQ ID NO:4) starting from any position between18 and 31 and ending at any position between 157 and 175 orcorresponding to residues of H19011_1_P8_V1 (SEQ ID NO:5) starting fromany position between 18 and 31 and ending at any position between 157and 175, or corresponding to residues of H19011_1_P9 (SEQ ID NO:6)starting from any position between 18 and 31 and ending at any positionbetween 159 and 172, or corresponding to residues of H19011_1_P9_V1 (SEQID NO:34) starting from any position between 18 and 31 and ending at anyposition between 159 and 172.

Generally, the C1ORF32 polypeptide fragments are expressed from nucleicacids that include sequences that encode a signal sequence. The signalsequence is generally cleaved from the immature polypeptide to producethe mature polypeptide lacking the signal sequence. The signal sequenceof C1ORF32 can be replaced by the signal sequence of another polypeptideusing standard molecule biology techniques to affect the expressionlevels, secretion, solubility, or other property of the polypeptide. Thesignal sequence that is used to replace the C1ORF32 signal sequence canbe any known in the art.

In a further embodiment, the fusion protein includes the extracellulardomain of C1ORF32, or a fragment thereof fused to an Ig Fc region.Recombinant IgC1ORF32 polypeptides, fragments or fusion proteins thereoffusion proteins can be prepared by fusing the coding region of theextracellular domain of C1ORF32 or a fragment thereof to the Fc regionof human IgG1 or mouse IgG2a, as described previously (Chapoval, et al.,Methods MoI. Med, 45:247-255 (2000)).

Variants of C1ORF32 Polypeptides

Useful variants of such C1ORF32 polypeptides include those that increasebiological activity, as indicated by any of the assays described herein,or that increase half life or stability of the protein. Soluble C1ORF32polypeptides and C1ORF32 fragments, or fusions thereof having C1ORF32activity, can be engineered to increase biological activity. In afurther embodiment, the C1ORF32 polypeptide or fusion protein has beenmodified with at least one amino acid substitution, deletion, orinsertion that increases the binding of the molecule to an immune cell,for example a T cell, and transmits an inhibitory signal into the Tcell. Other optional variants are those C1ORF32 polypeptides that areengineered to selectively bind to one type of T cell versus other immunecells. For example, the C1ORF32 polypeptide can be engineered to bindoptionally to Tregs, Th0, Th1, Th17, Th2 or Th22 cells. Preferentialbinding refers to binding that is at least 10%, 20%, 30%, 40%, 50%, 60%f 70%, 80%, 90%, 95%, or greater for one type of cell over another typeof cell. Still other variants of C1ORF32 can be engineered to havereduced binding to immune cells relative to wildtype C1ORF32. Thesevariants can be used in combination with variants having strongerbinding properties to modulate the immune response with a moderateimpact. Also optionally, soluble C1ORF32 polypeptides and C1ORF32fragments, or fusions thereof having C1ORF32 activity, can be engineeredto have an increased half-life relative to wildtype. These variantstypically are modified to resist enzymatic degradation. Exemplarymodifications include modified amino acid residues and modified peptidebonds that resist enzymatic degradation. Various modifications toachieve this are known in the art.

Also optionally, variant C1ORF32 polypeptides can be engineered toprevent a cleavage of the full C1ORF32 ECD (SEQ ID NO:14) between aminoacids F and A at positions 179 and 180 of H19011_1_P8_V1 or H19011_1_P8(Seq ID NOs: 4 or 5). According to at least some embodiments of thepresent invention there is provided one or more amino acid insertions,deletions or substitutions that prevent the cleavage of the C1ORF32polypeptides, fusion proteins, or fragments thereof. Suitable amino acidsubstitutions include conservative and non-conservative substitutions,as described above. According to at least some embodiments of thepresent invention the amino acid substitution in the cleavage site ofC1ORF32 ECD at positions 179 and 180 of H19011_1_P8_V1 or H19011_1_P8(Seq ID NOs: 4 or 5) is selected from but not limited to: FA->GA (as forexample disclosed in SEQ ID NO:64); FA->AA (as for example disclosed inSEQ ID NO:96); and FA->GG (as for example disclosed in SEQ ID NO:45).

The terms “individual”, “host”, “subject”, and “patient” are usedinterchangeably herein, and refer any human or nonhuman animal. The term“nonhuman animal” includes all vertebrates, e.g., mammals andnon-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cowschickens, amphibians, reptiles, etc.

Various aspects of the invention are described in further detail in thefollowing subsections.

Nucleic Acids

A “nucleic acid fragment” or an “oligonucleotide” or a “polynucleotide”are used herein interchangeably to refer to a polymer of nucleic acidresidues. A polynucleotide sequence of the present invention refers to asingle or double stranded nucleic acid sequences which is isolated andprovided in the form of an RNA sequence, a complementary polynucleotidesequence (cDNA), a genomic polynucleotide sequence and/or a compositepolynucleotide sequences (e.g., a combination of the above).

Thus, the present invention encompasses nucleic acid sequences describedhereinabove; fragments thereof, sequences hybridizable therewith,sequences homologous thereto [e.g., at least 90%, at least 95, 96, 97,98 or 99% or more identical to the nucleic acid sequences set forthherein, sequences encoding similar polypeptides with different codonusage, altered sequences characterized by mutations, such as deletion,insertion or substitution of one or more nucleotides, either naturallyoccurring or man induced, either randomly or in a targeted fashion. Thepresent invention also encompasses homologous nucleic acid sequences(i.e., which form a part of a polynucleotide sequence of the presentinvention), which include sequence regions unique to the polynucleotidesof the present invention.

In cases where the polynucleotide sequences of the present inventionencode previously unidentified polypeptides, the present invention alsoencompasses novel polypeptides or portions thereof, which are encoded bythe isolated polynucleotide and respective nucleic acid fragmentsthereof described hereinabove and/or degenerative variants thereof.

Thus, the present invention also encompasses polypeptides encoded by thepolynucleotide sequences of the present invention. The present inventionalso encompasses homologues of these polypeptides, such homologues canbe at least 90%, at least 95, 96, 97, 98 or 99% or more homologous tothe amino acid sequences set forth below, as can be determined usingBlastP software of the National Center of Biotechnology Information(NCBI) using default parameters.

Finally, the present invention also encompasses fragments of the abovedescribed polypeptides and polypeptides having mutations, such asdeletions, insertions or substitutions of one or more amino acids,either naturally occurring or man induced, either randomly or in atargeted fashion.

As mentioned hereinabove, biomolecular sequences of the presentinvention can be efficiently utilized as tissue or pathological markersand as putative drugs or drug targets for treating or preventing adisease.

Oligonucleotides designed for carrying out the methods of the presentinvention for any of the sequences provided herein (designed asdescribed above) can be generated according to any oligonucleotidesynthesis method known in the art such as enzymatic synthesis or solidphase synthesis. Equipment and reagents for executing solid-phasesynthesis are commercially available from, for example, AppliedBiosystems. Any other means for such synthesis may also be employed; theactual synthesis of the oligonucleotides is well within the capabilitiesof one skilled in the art.

Oligonucleotides used according to this aspect of the present inventionare those having a length selected from a range of about 10 to about 200bases preferably about 15 to about 150 bases, more preferably about 20to about 100 bases, most preferably about 20 to about 50 bases.

The oligonucleotides of the present invention may comprise heterocyclicnucleosides consisting of purines and the pyrimidines bases, bonded in a3′ to 5′ phosphodiester linkage.

Preferable oligonucleotides are those modified in either backbone,internucleoside linkages or bases, as is broadly described hereinunder.Such modifications can oftentimes facilitate oligonucleotide uptake andresistivity to intracellular conditions.

Specific examples of preferred oligonucleotides useful according to thisaspect of the present invention include oligonucleotides containingmodified backbones or non-natural internucleoside linkages.Oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone, as disclosed in U.S. Pat. Nos.4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466, 677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms can also be used.

Alternatively, modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts, as disclosed in U.S. Pat. Nos. 5,034,506;5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562;5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240;5,608,046; 5,610,289; 5,618,704; 5,623, 070; 5,663,312; 5,633,360;5,677,437; and 5,677,439.

Other oligonucleotides which can be used according to the presentinvention, are those modified in both sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for complementation with theappropriate polynucleotide target. An example for such anoligonucleotide mimetic, includes peptide nucleic acid (PNA). A PNAoligonucleotide refers to an oligonucleotide where the sugar-backbone isreplaced with an amide containing backbone, in particular anaminoethylglycine backbone. The bases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Other backbone modifications, which can be used in thepresent invention are disclosed in U.S. Pat. No. 6,303,374.

Oligonucleotides of the present invention may also include basemodifications or substitutions. As used herein, “unmodified” or“natural” bases include the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified bases include but are not limited to other synthetic andnatural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Further bases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science andEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Such bases areparticularly useful for increasing the binding affinity of theoligomeric compounds of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. [Sanghvi Y S et al. (1993) AntisenseResearch and Applications, CRC Press, Boca Raton 276-278] and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates, which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety, cholic acid,a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety, asdisclosed in U.S. Pat. No. 6,303,374.

It is not necessary for all positions in a given oligonucleotidemolecule to be uniformly modified, and in fact more than one of theaforementioned modifications may be incorporated in a single compound oreven at a single nucleoside within an oligonucleotide.

Peptides

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.Polypeptides can be modified, e.g., by the addition of carbohydrateresidues to form glycoproteins. The terms “polypeptide,” “peptide” and“protein” include glycoproteins, as well as non-glycoproteins.

Polypeptide products can be biochemically synthesized such as byemploying standard solid phase techniques. Such methods includeexclusive solid phase synthesis, partial solid phase synthesis methods,fragment condensation, classical solution synthesis. These methods arepreferably used when the peptide is relatively short (i.e., 10 kDa)and/or when it cannot be produced by recombinant techniques (i.e., notencoded by a nucleic acid sequence) and therefore involves differentchemistry.

Solid phase polypeptide synthesis procedures are well known in the artand further described by John Morrow Stewart and Janis Dillaha Young,Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Synthetic polypeptides can be purified by preparative high performanceliquid chromatography [Creighton T. (1983) Proteins, structures andmolecular principles. WH Freeman and Co. N.Y.] and the composition ofwhich can be confirmed via amino acid sequencing.

In cases where large amounts of a polypeptide are desired, it can begenerated using recombinant techniques such as described by Bitter etal., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990)Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514,Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J.3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al.(1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988,Methods for Plant Molecular Biology, Academic Press, NY, Section VIII,pp 421-463.

It will be appreciated that peptides identified according to theteachings of the present invention may be degradation products,synthetic peptides or recombinant peptides as well as peptidomimetics,typically, synthetic peptides and peptoids and semipeptoids which arepeptide analogs, which may have, for example, modifications renderingthe peptides more stable while in a body or more capable of penetratinginto cells. Such modifications include, but are not limited to Nterminus modification, C terminus modification, peptide bondmodification, including, but not limited to, CH2-NH, CH2-S, CH2-S═O,O═C—NH, CH2-O, CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications,and residue modification. Methods for preparing peptidomimetic compoundsare well known in the art and are specified, for example, inQuantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. ChoplinPergamon Press (1992), which is incorporated by reference as if fullyset forth herein. Further details in this respect are providedhereinunder.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH3)-CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2-), α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH2-NH—), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted bysynthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine(Nol), ring-methylated derivatives of Phe, halogenated derivatives ofPhe or o-methyl-Tyr.

In addition to the above, the peptides of the present invention may alsoinclude one or more modified amino acids or one or more non-amino acidmonomers (e.g. fatty acids, complex carbohydrates etc).

As used herein in the specification and in the claims section below theterm “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids.

Since the peptides of the present invention are preferably utilized intherapeutics which require the peptides to be in soluble form, thepeptides of the present invention preferably include one or morenon-natural or natural polar amino acids, including but not limited toserine and threonine which are capable of increasing peptide solubilitydue to their hydroxyl-containing side chain.

In cases where large amounts of the peptides of the present inventionare desired, the peptides of the present invention can be generatedusing recombinant techniques such as described by Bitter et al., (1987)Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods inEnzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsuet al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J.3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al.(1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988,Methods for Plant Molecular Biology, Academic Press, NY, Section VIII,pp 421-463.

Expression Systems

To enable cellular expression of the polynucleotides of the presentinvention, a nucleic acid construct according to the present inventionmay be used, which includes at least a coding region of one of the abovenucleic acid sequences, and further includes at least one cis actingregulatory element. As used herein, the phrase “cis acting regulatoryelement” refers to a polynucleotide sequence, preferably a promoter,which binds a trans acting regulator and regulates the transcription ofa coding sequence located downstream thereto.

Any suitable promoter sequence can be used by the nucleic acid constructof the present invention.

Preferably, the promoter utilized by the nucleic acid construct of thepresent invention is active in the specific cell population transformed.Examples of cell type-specific and/or tissue-specific promoters includepromoters such as albumin that is liver specific [Pinkert et al., (1987)Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al.,(1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cellreceptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins;[Banerji et al. (1983) Cell 33729-740], neuron-specific promoters suchas the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad.Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al.(1985) Science 230:912-916] or mammary gland-specific promoters such asthe milk whey promoter (U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). The nucleic acid construct of the presentinvention can further include an enhancer, which can be adjacent ordistant to the promoter sequence and can function in up regulating thetranscription therefrom.

The nucleic acid construct of the present invention preferably furtherincludes an appropriate selectable marker and/or an origin ofreplication. Preferably, the nucleic acid construct utilized is ashuttle vector, which can propagate both in E. coli (wherein theconstruct comprises an appropriate selectable marker and origin ofreplication) and be compatible for propagation in cells, or integrationin a gene and a tissue of choice. The construct according to the presentinvention can be, for example, a plasmid, a bacmid, a phagemid, acosmid, a phage, a virus or an artificial chromosome.

Examples of suitable constructs include, but are not limited to, pcDNA3,pcDNA3.1 (+/−), pGL3, PzeoSV2 (+/−), pDisplay, pEF/myc/cyto,pCMV/myc/cyto each of which is commercially available from InvitrogenCo. (www.invitrogen.com). Examples of retroviral vector and packagingsystems are those sold by Clontech, San Diego, Calif., including Retro-Xvectors pLNCX and pLXSN, which permit cloning into multiple cloningsites and the transgene is transcribed from CMV promoter. Vectorsderived from Mo-MuLV are also included such as pBabe, where thetransgene will be transcribed from the 5′LTR promoter.

Currently preferred in vivo nucleic acid transfer techniques includetransfection with viral or non-viral constructs, such as adenovirus,lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) andlipid-based systems. Useful lipids for lipid-mediated transfer of thegene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al.,Cancer Investigation, 14(1): 54-65 (1996)]. The most preferredconstructs for use in gene therapy are viruses, most preferablyadenoviruses, AAV, lentiviruses, or retroviruses. A viral construct suchas a retroviral construct includes at least one transcriptionalpromoter/enhancer or locus-defining elements, or other elements thatcontrol gene expression by other means such as alternate splicing,nuclear RNA export, or post-translational modification of messenger.Such vector constructs also include a packaging signal, long terminalrepeats (LTRs) or portions thereof, and positive and negative strandprimer binding sites appropriate to the virus used, unless it is alreadypresent in the viral construct. In addition, such a construct typicallyincludes a signal sequence for secretion of the peptide from a host cellin which it is placed. Preferably the signal sequence for this purposeis a mammalian signal sequence or the signal sequence of thepolypeptides of the present invention. Optionally, the construct mayalso include a signal that directs polyadenylation, as well as one ormore restriction sites and a translation termination sequence. By way ofexample, such constructs will typically include a 5′ LTR, a tRNA bindingsite, a packaging signal, an origin of second-strand DNA synthesis, anda 3′ LTR or a portion thereof. Other vectors can be used that arenon-viral, such as cationic lipids, polylysine, and dendrimers.

Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a protein of theinvention, or derivatives, fragments, analogs or homologs thereof. Asused herein, the term “vector” refers to a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked. Onetype of vector is a “plasmid”, which refers to a circular doublestranded DNA loop into which additional DNA segments can be ligated.Another type of vector is a viral vector, wherein additional DNAsegments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively-linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably-linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequences in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell).

The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include those thatdirect constitutive expression of a nucleotide sequence in many types ofhost cell and those that direct expression of the nucleotide sequenceonly in certain host cells (e.g., tissue-specific regulatory sequences).It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. The expression vectors of the invention can be introduced into hostcells to thereby produce proteins or peptides, including fusion proteinsor peptides, encoded by nucleic acids as described herein.

The recombinant expression vectors of the invention can be designed forproduction of variant proteins in prokaryotic or eukaryotic cells. Forexample, proteins of the invention can be expressed in bacterial cellssuch as Escherichia coli, insect cells (using baculovirus expressionvectors) yeast cells or mammalian cells. Suitable host cells arediscussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out inEscherichia coli with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionproteins. Fusion vectors add a number of amino acids to a proteinencoded therein, to the amino or C terminus of the recombinant protein.Such fusion vectors typically serve three purposes: (i) to increaseexpression of recombinant protein; (ii) to increase the solubility ofthe recombinant protein; and (iii) to aid in the purification of therecombinant protein by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantprotein to enable separation of the recombinant protein from the fusionmoiety subsequent to purification of the fusion protein. Such enzymes,and their cognate recognition sequences, include Factor Xa, thrombin,PreScission, TEV and enterokinase. Typical fusion expression vectorsinclude pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRITS (Pharmacia,Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89)—not accurate, pET11a-dhave N terminal T7 tag.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacterium with an impaired capacity toproteolytically cleave the recombinant protein. See, e.g., Gottesman,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990) 119-128. Another strategy is to alter thenucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (see, e.g., Wada, et al., 1992.Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acidsequences of the invention can be carried out by standard DNA synthesistechniques. Another strategy to solve codon bias is by using BL21-codonplus bacterial strains (Invitrogen) or Rosetta bacterial strain(Novagen), these strains contain extra copies of rare E. coli tRNAgenes.

In another embodiment, the expression vector encoding for the protein ofthe invention is a yeast expression vector. Examples of vectors forexpression in yeast Saccharomyces cerevisiae include pYepSec1 (Baldari,et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982.Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123),pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogenCorp, San Diego, Calif.).

Alternatively, polypeptides of the present invention can be produced ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840)and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195), pIRESpuro(Clontech), pUB6 (Invitrogen), pCEP4 (Invitrogen) pREP4 (Invitrogen),pcDNA3 (Invitrogen). When used in mammalian cells, the expressionvector's control functions are often provided by viral regulatoryelements. For example, commonly used promoters are derived from polyoma,adenovirus 2, cytomegalovirus, Rous Sarcoma Virus, and simian virus 40.For other suitable expression systems for both prokaryotic andeukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the alpha-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546).

The present invention in at least some embodiments further provides arecombinant expression vector comprising a DNA molecule of the inventioncloned into the expression vector in an antisense orientation. That is,the DNA molecule is operatively-linked to a regulatory sequence in amanner that allows for expression (by transcription of the DNA molecule)of an RNA molecule that is antisense to mRNA encoding for protein of theinvention. Regulatory sequences operatively linked to a nucleic acidcloned in the antisense orientation can be chosen that direct thecontinuous expression of the antisense RNA molecule in a variety of celltypes, for instance viral promoters and/or enhancers, or regulatorysequences can be chosen that direct constitutive, tissue specific orcell type specific expression of antisense RNA. The antisense expressionvector can be in the form of a recombinant plasmid, phagemid orattenuated virus in which antisense nucleic acids are produced under thecontrol of a high efficiency regulatory region, the activity of whichcan be determined by the cell type into which the vector is introduced.For a discussion of the regulation of gene expression using antisensegenes see, e.g., Weintraub, et al., “Antisense RNA as a molecular toolfor genetic analysis,” Reviews-Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example,protein of the invention can be produced in bacterial cells such as E.coli, insect cells, yeast, plant or mammalian cells (such as Chinesehamster ovary cells (CHO) or COS or 293 cells). Other suitable hostcells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin, puromycin, blasticidin and methotrexate. Nucleicacids encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding protein of the invention or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) protein of theinvention. Accordingly, the present invention in at least someembodiments further provides methods for producing proteins of theinvention using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of the present invention (intowhich a recombinant expression vector encoding protein of the inventionhas been introduced) in a suitable medium such that the protein of theinvention is produced. In another embodiment, the method furthercomprises isolating protein of the invention from the medium or the hostcell.

For efficient production of the protein, it is preferable to place thenucleotide sequences encoding the protein of the invention under thecontrol of expression control sequences optimized for expression in adesired host. For example, the sequences may include optimizedtranscriptional and/or translational regulatory sequences (such asaltered Kozak sequences).

It should be noted, that according to at least some embodiments of thepresent invention the C1ORF32 polypeptides as described herein mayoptionally be isolated as naturally-occurring polypeptides, or from anysource whether natural, synthetic, semi-synthetic or recombinant.Accordingly, the C1ORF32 proteins may be isolated as naturally-occurringproteins from any species, particularly mammalian, including bovine,ovine, porcine, murine, equine, and preferably human. Alternatively, theC1ORF32 proteins may be isolated as recombinant polypeptides that areexpressed in prokaryote or eukaryote host cells, or isolated as achemically synthesized polypeptide.

A skilled artisan can readily employ standard isolation methods toobtain isolated C1ORF32 proteins. The nature and degree of isolationwill depend on the source and the intended use of the isolatedmolecules.

Fusion Proteins

According to at least some embodiments, C1ORF32 fusion polypeptides havea first fusion partner comprising all or a part of a C1ORF32 proteinfused to a second polypeptide directly or via a linker peptide sequenceor a chemical linker useful to connect the two proteins. The C1ORF32polypeptide may or may not contain the native signal peptide. TheC1ORF32 polypeptide may optionally be fused to a second polypeptide toform a fusion protein as described herein. The presence of the secondpolypeptide can alter the solubility, stability, affinity and/or valencyof the C1ORF32 fusion polypeptide. As used herein, “valency” refers tothe number of binding sites available per molecule. In one embodimentthe second polypeptide is a polypeptide from a different source ordifferent protein.

According to at least some embodiments, the C1ORF32 protein or fragmentis selected for its activity for the treatment of immune relateddisorder and/or infection and/or according to one or more in vitrobiological activities as described herein.

In one embodiment, the second polypeptide contains one or more domainsof an immunoglobulin heavy chain constant region, preferably having anamino acid sequence corresponding to the hinge, CH2 and CH3 regions of ahuman immunoglobulin Cγ1, Cγ2, Cγ3 or Cγ4, chains or to the hinge, CH2and CH3 regions of a murine immunoglobulin Cγ2a chain. SEQ ID NO: 20provides exemplary sequence for the hinge, CH2 and CH3 regions of ahuman immunoglobulin Cγ1.

According to at least some embodiments, the fusion protein is a dimericfusion protein. In an optional dimeric fusion protein, the dimer resultsfrom the covalent bonding of Cys residue in the hinge region of two ofthe Ig heavy chains that are the same Cys residues that are disulfidelinked in dimerized normal Ig heavy chains. Such proteins are referredto as IgC1ORF32 polypeptides, fragments or fusion proteins thereof.

In one embodiment, the immunoglobulin constant domain may contain one ormore amino acid insertions, deletions or substitutions that enhancebinding to specific cell types, increase the bioavailablity, or increasethe stability of the C1ORF32 polypeptides, fusion proteins, or fragmentsthereof. Suitable amino acid substitutions include conservative andnon-conservative substitutions, as described above.

The fusion proteins optionally contain a domain that functions todimerize or multimerize two or more fusion proteins. Thepeptide/polypeptide linker domain can either be a separate domain, oralternatively can be contained within one of the other domains (C1ORF32polypeptide or second polypeptide) of the fusion protein. Similarly, thedomain that functions to dimerize or multimerize the fusion proteins caneither be a separate domain, or alternatively can be contained withinone of the other domains (C1ORF32 polypeptide, second polypeptide orpeptide/polypeptide linker domain) of the fusion protein. In oneembodiment, the dimerization/multimerization domain and thepeptide/polypeptide linker domain are the same. Further specific,illustrative and non-limiting examples of dimerization/multimerizationdomains and linkers are given below.

Fusion proteins disclosed herein according to at least some embodimentsof the present invention are of formula I: N-R1-R2-R3-C wherein “N”represents the N-terminus of the fusion protein, “C” represents theC-terminus of the fusion protein. In the further embodiment, “R1” is aC1ORF32 polypeptide, “R2” is an optional peptide/polypeptide or chemicallinker domain, and “R3” is a second polypeptide. Alternatively, R3 maybe a C1ORF32 polypeptide and R1 may be a second polypeptide. Variousnon-limiting examples of linkers are described in greater detail below.

Optionally, the fusion protein comprises the C1ORF32 polypeptidefragments selected from the group consisting of SEQ ID NOs: 29, 30,41-105, fused, optionally by a linker peptide of one or more amino acids(e.g. GS) to one or more “half-life extending moieties”. A “half-lifeextending moiety” is any moiety, for example, a polypeptide, smallmolecule or polymer, that, when appended to protein, extends the in vivohalf-life of that protein in the body of a subject (e.g., in the plasmaof the subject). For example, a half-life extending moiety is, in anembodiment of the invention, polyethylene glycol (PEG), monomethoxy PEG(mPEG) or an immunoglobulin (Ig). In an embodiment of the invention, PEGis a 5, 10, 12, 20, 30, 40 or 50 kDa moiety or larger or comprises about12000 ethylene glycol units (PEG12000).

The fusion protein may also optionally be prepared by chemical syntheticmethods and the “join” effected chemically, either during synthesis orpost-synthesis. Cross-linking and other such methods may optionally beused (optionally also with the above described genetic level fusionmethods), as described for example in U.S. Pat. No. 5,547,853 to Wallneret al, which is hereby incorporated by reference as if fully set forthherein as a non-limiting example only.

According to the present invention, a fusion protein may be preparedfrom a protein of the invention by fusion with a portion of animmunoglobulin comprising a constant region of an immunoglobulin. Morepreferably, the portion of the immunoglobulin comprises a heavy chainconstant region which is optionally and more preferably a human heavychain constant region. The heavy chain constant region is mostpreferably an IgG heavy chain constant region, and optionally and mostpreferably is an Fc chain, most preferably an IgG Fc fragment thatcomprises the hinge, CH2 and CH3 domains. The Fc chain may optionally bea known or “wild type” Fc chain, or alternatively may be mutated ortruncated. The Fc portion of the fusion protein may optionally be variedby isotype or subclass, may be a chimeric or hybrid, and/or may bemodified, for example to improve effector functions, control ofhalf-life, tissue accessibility, augment biophysical characteristicssuch as stability, and improve efficiency of production (and lesscostly). Many modifications useful in construction of disclosed fusionproteins and methods for making them are known in the art, see forexample Mueller, et al, MoI. Immun., 34(6):441-452 (1997), Swann, etal., Cur. Opin. Immun., 20:493-499 (2008), and Presta, Cur. Opin. Immun20:460-470 (2008). In some embodiments the Fc region is the native IgG1,IgG2, or IgG4 Fc region. In some embodiments the Fc region is a hybrid,for example a chimeric consisting of IgG2/IgG4 Fc constant regions.

Modications to the Fc region include, but are not limited to, IgG4modified to prevent binding to Fc gamma receptors and complement, IgG1modified to improve binding to one or more Fc gamma receptors, IgG1modified to minimize effector function (amino acid changes), IgG1 withaltered/no glycan (typically by changing expression host or substutingthe Asn at position 297), and IgG1 with altered pH-dependent binding toFcRn. The Fc region may include the entire hinge region, or less thanthe entire hinge region.

In another embodiment, the Fc domain may contain one or more amino acidinsertions, deletions or substitutions that reduce binding to the lowaffinity inhibitory Fc receptor CD32B (FcγRIIB) and retain wild-typelevels of binding to or enhance binding to the low affinity activatingFc receptor CD16A (FcγRIIIA).

Another embodiment includes IgG2-4 hybrids and IgG4 mutants that havereduced binding to FcR (Fc receptor) which increase their half life.Representative IgG2-4 hybrids and IgG4 mutants are described in Angal,S. et al., Molecular Immunology, 30(1):105-108 (1993); Mueller, J. etal., Molecular Immunology, 34(6): 441-452 (1997); and U.S. Pat. No.6,982,323 to Wang et al. In some embodiments the IgG1 and/or IgG2 domainis deleted; for example, Angal et al. describe IgG1 and IgG2 havingserine 241 replaced with a proline.

In a further embodiment, the Fc domain contains amino acid insertions,deletions or substitutions that enhance binding to CD16A. A large numberof substitutions in the Fc domain of human IgG1 that increase binding toCD16A and reduce binding to CD32B are known in the art and are describedin Stavenhagen, et al., Cancer Res., 57(18):8882-90 (2007). Exemplaryvariants of human IgG1 Fc domains with reduced binding to CD32B and/orincreased binding to CD16A contain F243L, R929P, Y300L, V305I or P296Lsubstitutions. These amino acid substitutions may be present in a humanIgG1 Fc domain in any combination.

In one embodiment, the human IgG1 Fc domain variant contains a F243L,R929P and Y300L substitution. In another embodiment, the human IgG1 Fcdomain variant contains a F243L, R929P, Y300L, V3O5I and P296Lsubstitution. In another embodiment, the human IgG1 Fc domain variantcontains an N297A/Q substitution, as these mutations abolishFcγRbinding. Non-limiting, illustrative, exemplary types of mutations aredescribed in US Patent Application No. 20060034852, published on Feb.16, 2006, hereby incorporated by reference as if fully set forth herein.The term “Fc chain” also optionally comprises any type of Fc fragment.

Several of the specific amino acid residues that are important forantibody constant region-mediated activity in the IgG subclass have beenidentified. Inclusion, substitution or exclusion of these specific aminoacids therefore allows for inclusion or exclusion of specificimmunoglobulin constant region-mediated activity. Furthermore, specificchanges may result in aglycosylation for example and/or other desiredchanges to the Fc chain. At least some changes may optionally be made toblock a function of Fc which is considered to be undesirable, such as anundesirable immune system effect, as described in greater detail below.

Non-limiting, illustrative examples of mutations to Fc which may be madeto modulate the activity of the fusion protein include the followingchanges (given with regard to the Fc sequence nomenclature as given byKabat, from Kabat E A et al: Sequences of Proteins of ImmunologicalInterest. US Department of Health and Human Services, NIH, 1991):220C->S; 233-238 ELLGGP->EAEGAP; 265D->A, preferably in combination with434N->A; 297N->A (for example to block N-glycosylation); 318-322EYKCK->AYACA; 330-331AP->SS; or a combination thereof (see for exampleM. Clark, “Chemical Immunol and Antibody Engineering”, pp 1-31 for adescription of these mutations and their effect). The construct for theFc chain which features the above changes optionally and preferablycomprises a combination of the hinge region with the CH2 and CH3domains.

The above mutations may optionally be implemented to enhance desiredproperties or alternatively to block non-desired properties. Forexample, aglycosylation of antibodies was shown to maintain the desiredbinding functionality while blocking depletion of T-cells or triggeringcytokine release, which may optionally be undesired functions (see M.Clark, “Chemical Immunol and Antibody Engineering”, pp 1-31).Substitution of 331proline for serine may block the ability to activatecomplement, which may optionally be considered an undesired function(see M. Clark, “Chemical Immunol and Antibody Engineering”, pp 1-31).Changing 330 alanine to serine in combination with this change may alsoenhance the desired effect of blocking the ability to activatecomplement.

Residues 235 and 237 were shown to be involved in antibody-dependentcell-mediated cytotoxicity (ADCC), such that changing the block ofresidues from 233-238 as described may also block such activity if ADCCis considered to be an undesirable function.

Residue 220 is normally a cysteine for Fc from IgG1, which is the siteat which the heavy chain forms a covalent linkage with the light chain.Optionally, this residue may be changed to another amino acid residue(e.g., serine), to avoid any type of covalent linkage (see M. Clark,“Chemical Immunol and Antibody Engineering”, pp 1-31) or by deletion ortruncation.

The above changes to residues 265 and 434 may optionally be implementedto reduce or block binding to the Fc receptor, which may optionallyblock undesired functionality of Fc related to its immune systemfunctions (see “Binding site on Human IgG1 for Fc Receptors”, Shields etal, Vol 276, pp 6591-6604, 2001).

The above changes are intended as illustrations only of optional changesand are not meant to be limiting in any way. Furthermore, the aboveexplanation is provided for descriptive purposes only, without wishingto be bound by a single hypothesis.

In a further embodiment, the fusion protein includes the C1ORF32fragment fused to an Ig Fc region. Recombinant Ig-C1ORF32 fragmentpolypeptides can be prepared by fusing the coding region of the C1ORF32fragment to the Fc region of human IgG1 or mouse IgG2a, as describedpreviously (Chapoval, et al., Methods MoI. Med, 45:247-255 (2000)).Optionally, C1ORF32 fusion protein, comprising an amino acid sequence ofhuman C1ORF32 ECD fragment fused to murine or human immunoglobulin Fc.Optionally, said fusion protein comprises the amino acid sequence setforth in anyone of SEQ ID NOs: 29, 30, 41-105, and/or 45, 64, or 96,fused to human IgG1 Fc set forth in any one of SEQ ID NOs: 20, 21, 115.Optionally, the amino acid sequence of said fusion protein is set forthin SEQ ID NO: 39, 108-112, 116-190; optionally and preferably, the aminoacid sequence is set forth in any of SEQ ID NOs:112, 120 oralternatively in any one of SEQ ID NOs:110, 136.

The aforementioned exemplary fusion proteins can incorporate anycombination of the variants described herein. In another embodiment theterminal lysine of the aforementioned exemplary fusion proteins isdeleted.

The disclosed fusion proteins can be isolated using standard molecularbiology techniques. For example, an expression vector containing a DNAsequence encoding a C1ORF32 ECD fragments or fusion proteins thereoffusion protein is transfected into 293 cells by calcium phosphateprecipitation and cultured in serum-free DMEM. The supernatant iscollected at 72 h and the fusion protein is purified by Protein G, orpreferably Protein A SEPHAROSE® columns (Pharmacia, Uppsala, Sweden).Optionally, a DNA sequence encoding a C1ORF32 fragments fusion proteinis transfected into GPEx® retrovectors and expressed in CHO—S cellsfollowing four rounds of retrovector transduction. The protein isclarified from supernatants using protein A chromatography.

In another embodiment the second polypeptide may have a conjugationdomain through which additional molecules can be bound to the C1ORF32fragments fusion proteins. In one such embodiment, the conjugatedmolecule is capable of targeting the fusion protein to a particularorgan or tissue; further specific, illustrative, non-limiting examplesof such targeting domains and/or molecules are given below.

In another such embodiment the conjugated molecule is anotherimmunomodulatory agent that can enhance or augment the effects of theC1ORF32 fusion protein. In another embodiment the conjugated molecule isPolyethylene Glycol (PEG).

Peptide or Polypeptide Linker Domain

The disclosed C1ORF32 fusion proteins optionally contain a peptide orpolypeptide linker domain that separates the C1ORF32 polypeptide fromthe second polypeptide. In one embodiment, the linker domain containsthe hinge region of an immunoglobulin. In a further embodiment, thehinge region is derived from a human immunoglobulin. Suitable humanimmunoglobulins that the hinge can be derived from include IgG, IgD andIgA. In a further embodiment, the hinge region is derived from humanIgG. Amino acid sequences of immunoglobulin hinge regions and otherdomains are well known in the art. In one embodiment, C1ORF32 fusionpolypeptides contain the hinge, CH2 and CH3 regions of a humanimmunoglobulin Cγ1 chain, optionally with the Cys at position 220(according to full length human IgG1, position 5 in SEQ ID NO: 20)replaced with a Ser (SEQ ID NO: 115) having at least 85%, 90%, 95%, 99%or 100% sequence homology to amino acid sequence set forth in SEQ ID NO:20:

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKThe hinge can be further shortened to remove amino acids 1, 2, 3, 4, 5,or combinations thereof of any one of SEQ ID NOs: 20 or 115. In oneembodiment, amino acids 1-5 of any one of SEQ ID NOs: 20 or 115 aredeleted.

In another embodiment, C1ORF32 fusion polypeptides contain the CH2 andCH3 regions of a human immunoglobulin Cγ1 chain having at least 85%,90%, 95%, 99% or 100% sequence homology to amino acid sequence set forthin SEQ ID NO: 21:

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGKIn another embodiment, the C1ORF32 fusion polypeptides contain the CH2and CH3 regions of a murine immunoglobulin Cγ2a chain at least 85%, 90%,95%, 99% or 100% sequence homology to amino acid sequence set forth inSEQ ID NO:31:

EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.In another embodiment, the linker domain contains a hinge region of animmunoglobulin as described above, and further includes one or moreadditional immunoglobulin domains.

Other suitable peptide/polypeptide linker domains include naturallyoccurring or non-naturally occurring peptides or polypeptides. Peptidelinker sequences are at least 2 amino acids in length. Optionally thepeptide or polypeptide domains are flexible peptides or polypeptides. A“flexible linker” herein refers to a peptide or polypeptide containingtwo or more amino acid residues joined by peptide bond(s) that providesincreased rotational freedom for two polypeptides linked thereby thanthe two linked polypeptides would have in the absence of the flexiblelinker. Such rotational freedom allows two or more antigen binding sitesjoined by the flexible linker to each access target antigen(s) moreefficiently. Exemplary flexible peptides/polypeptides include, but arenot limited to, the amino acid sequences Gly-Ser (SEQ ID NO: 24),Gly-Ser-Gly-Ser (SEQ ID NO: 25), Ala-Ser (SEQ ID NO:26), Gly-Gly-Gly-Ser(SEQ ID NO: 27), Gly4-Ser (SEQ ID NO: 106), (Gly4-Ser)2 (SEQ ID NO:107), (Gly4-Ser)3 (SEQ ID NO: 32) and (Gly4-Ser)4 (SEQ ID NO: 33).Additional flexible peptide/polypeptide sequences are well known in theart. Other suitable peptide linker domains include helix forming linkerssuch as Ala-(Glu-Ala-Ala-Ala-Lys)n-Ala (n=1-5) (for n=1, SEQ ID NO:114as a non-limiting example). Additional helix forming peptide/polypeptidesequences are well known in the art. Additional example of a cleavablelinker is TEV-linker Gly Ser Glu Asn Leu Tyr Phe Gln Gly Ser Gly (SEQ IDNO:113). Non-limiting examples of C1ORF32 polypeptide fragments fused toFc portion via a linker are depicted in SEQ ID NOs: 8 and 108-112.

Dimerization, Multimerization and Targeting Domains

The fusion proteins disclosed herein optionally contain a dimerizationor multimerization domain that functions to dimerize or multimerize twoor more fusion proteins. The domain that functions to dimerize ormultimerize the fusion proteins can either be a separate domain, oralternatively can be contained within one of the other domains (C1ORF32polypeptide, second polypeptide, or peptide/polypeptide linker domain)of the fusion protein.

Dimerization or multinierization can occur between or among two or morefusion proteins through dimerization or multimerization domains.Alternatively, dimerization or multimerization of fusion proteins canoccur by chemical crosslinking. The dimers or multimers that are formedcan be homodimeric/homomultimeric or heterodimeric/heteromultimeric. Thesecond polypeptide “partner” in the C1ORF32 fusion polypeptides may becomprised of one or more other proteins, protein fragments or peptidesas described herein, including but not limited to any immunoglobulin(Ig) protein or portion thereof, preferably the Fc region, or a portionof a biologically or chemically active protein such as thepapillomavirus E7 gene product, melanoma-associated antigen p97), andHIV env protein (gp120). The “partner” is optionally selected to providea soluble dimer/multimer and/or for one or more other biologicalactivities as described herein.

A “dimerization domain” is formed by the association of at least twoamino acid residues or of at least two peptides or polypeptides (whichmay have the same, or different, amino acid sequences). The peptides orpolypeptides may interact with each other through covalent and/ornon-covalent associations). Optional dimerization domains contain atleast one cysteine that is capable of forming an intermoleculardisulfide bond with a cysteine on the partner fusion protein. Thedimerization domain can contain one or more cysteine residues such thatdisulfide bond(s) can form between the partner fusion proteins. In oneembodiment, dimerization domains contain one, two or three to about tencysteine residues. In a further embodiment, the dimerization domain isthe hinge region of an immunoglobulin.

Additional exemplary dimerization domains can be any known in the artand include, but not limited to, coiled coils, acid patches, zincfingers, calcium hands, a C_(H)1-C_(L) pair, an “interface” with anengineered “knob” and/or “protruberance” as described in U.S. Pat. No.5,821,333, leucine zippers (e.g., from jun and/or fos) (U.S. Pat. No.5,932,448), and/or the yeast transcriptional activator GCN4, SH2 (srchomology 2), SH3 (src Homology 3) (Vidal, et al, Biochemistry, 43,7336-44 ((2004)), phosphotyrosine binding (PTB) (Zhou, et al., Nature,378:584-592 (1995)), WW (Sudol, Prog, Biochys. MoL Bio., 65:113-132(1996)), PDZ (Kim, et al., Nature, 378: 85-88 (1995); Komau, et al,Science, 269.1737-1740 (1995)) 14-3-3, WD40 (Hu5 et al., J Biol Chem.,273, 33489-33494 (1998)) EH, Lim, an isoleucine zipper, a receptor dimerpair (e.g., interleukin-8 receptor (IL-8R); and integrin heterodimerssuch as LFA-I and GPIIIb/IIIa), or the dimerization region(s) thereof,dimeric ligand polypeptides (e.g. nerve growth factor (NGF),neurotrophin-3 (NT-3), interleukin-8 (IL-8), vascular endothelial growthfactor (VEGF), VEGF-C, VEGF-D, PDGF members, and brain-derivedneurotrophic factor (BDNF) (Arakawa, et al., J Biol. Chem., 269(45):27833-27839 (1994) and Radziejewski, et al., Biochem., 32(48): 1350(1993)) and can also be variants of these domains in which the affinityis altered. The polypeptide pairs can be identified by methods known inthe art, including yeast two hybrid screens. Yeast two hybrid screensare described in U.S. Pat. Nos. 5,283,173 and 6,562,576. Affinitiesbetween a pair of interacting domains can be determined using methodsknown in the art, including as described in Katahira, et at, J. BiolChem, 277, 9242-9246 (2002)). Alternatively, a library of peptidesequences can be screened for heterodimerization, for example, using themethods described in WO 01/00814. Useful methods for protein-proteininteractions are also described in U.S. Pat. No. 6,790,624.

A “multimerization domain” is a domain that causes three or morepeptides or polypeptides to interact with each other through covalentand/or non-covalent association(s). Suitable multimerization domainsinclude, but are not limited to, coiled-coil domains. A coiled-coil is apeptide sequence with a contiguous pattern of mainly hydrophobicresidues spaced 3 and 4 residues apart, usually in a sequence of sevenamino acids (heptad repeat) or eleven amino acids (undecad repeat),which assembles (folds) to form a multimeric bundle of helices.Coiled-coils with sequences including some irregular distribution of the3 and 4 residues spacing are also contemplated. Hydrophobic residues arein particular the hydrophobic amino acids Val, Ile, Leu, Met, Tyr, Pheand Trp. “Mainly hydrophobic” means that at least 50% of the residuesmust be selected from the mentioned hydrophobic amino acids.

The coiled coil domain may be derived from laminin. In the extracellularspace, the heterotrimeric coiled coil protein laminin plays an importantrole in the formation of basement membranes. Apparently, themultifunctional oligomeric structure is required for laminin function.Coiled coil domains may also be derived from the thrombospondins inwhich three (TSP-I and TSP-2) or five (TSP-3, TSP-4 and TSP-5) chainsare connected, or from COMP (COMPcc) (Guo, et at., EMBO J, 1998, 17:5265-5272) which folds into a parallel five-stranded coiled coil(Malashkevich, et al., Science, 274: 761-765 (1996)). Additional nonlimiting examples of coiled-coil domains derived from other proteins,and other domains that mediate polypeptide multimerization are known inthe art such as the vasodialator-stimulated phosphoprotein (VASP)domain, matrilin-1 (CMP), viral fusion peptides, soluble NSF(N-ethylmaleimide-sensitive factor) Attachment Protein receptor (SNARE)complexes, leucine-rich repeats, certain tRNA synthetases, are suitablefor use in the disclosed fusion proteins.

In another embodiment, C1ORF32 polypeptides, fusion proteins, orfragments thereof can be induced to form multimers by binding to asecond multivalent polypeptide, such as an antibody. Antibodies suitablefor use to multimerize C1ORF32 polypeptides, fusion proteins, orfragments thereof include, but are not limited to, IgM antibodies andcross-linked, multivalent IgG, IgA, IgD, or IgE complexes.

Dimerization or multimerization can occur between or among two or morefusion proteins through dimerization or multimerization domains,including those described above. Alternatively, dimerization ormultimerization of fusion proteins can occur by chemical crosslinking.Fusion protein dimers can be homodimers or heterodimers. Fusion proteinmultimers can be homomultimers or heteromultimers. Fusion protein dimersas disclosed herein are of formula N-R1-R2-R3-C N-R4-R5-R6-C or,alternatively, are of formula N-R1-R2-R3-C C-R4-R5-R6-N wherein thefusion proteins of the dimer provided by formula II are defined as beingin a parallel orientation and the fusion proteins of the dimer providedby formula III are defined as being in an antiparallel orientation.Parallel and antiparallel dimers are also referred to as cis and transdimers, respectively. “N” and “C” represent the N- and C-termini of thefusion protein, respectively. The fusion protein constituents “R1”, “R2”and “R3” are as defined above with respect to formula I. With respect toboth formula II and formula III, “R4” is a C1ORF32 polypeptide or asecond polypeptide, “R5” is an optional peptide/polypeptide linkerdomain, and “R6” is a C1ORF32 polypeptide or a second polypeptide,wherein “R6” is a C1ORF32 polypeptide when “R4” is a second polypeptide,and “R6′” is a second polypeptide when “R4” is a C1ORF32 polypeptide. Inone embodiment, “R1” is a C1ORF32 polypeptide, “R4” is also a C1ORF32polypeptide, and “R3” and “R6” are both second polypeptides.

Fusion protein dimers of formula II are defined as homodimers when“R1”=“R4”, “R2”=“R5” and “R3”=“R6”. Similarly, fusion protein dimers offormula III are defined as homodimers when “R1”=“R6”, “R2”=“R5” and“R3”=“R4”. Fusion protein dimers are defined as heterodimers when theseconditions are not met for any reason. For example, heterodimers maycontain domain orientations that meet these conditions (i.e., for adimer according to formula II, “R1” and “R4” are both C1ORF32polypeptides, “R2” and “R5” are both peptide/polypeptide linker domainsand “R3” and “R6” are both second polypeptides), however the species ofone or more of these domains is not identical. For example, although“R3” and “R6” may both be C1ORF32 polypeptides, one polypeptide maycontain a wild-type C1ORF32 amino acid sequence while the otherpolypeptide may be a variant C1ORF32 polypeptide. An exemplary variantC1ORF32 polypeptide is C1ORF32 polypeptide that has been modified tohave increased or decreased binding to a target cell, increased activityon immune cells, increased or decreased half life or stability. Dimersof fusion proteins that contain either a CHI or CL region of animmunoglobulin as part of the polypeptide linker domain preferably formheterodimers wherein one fusion protein of the dimer contains a CHIregion and the other fusion protein of the dimer contains a CL region.

Fusion proteins can also be used to form multimers. As with dimers,multimers may be parallel multimers, in which all fusion proteins of themultimer are aligned in the same orientation with respect to their N-and C-termini. Multimers may be antiparallel multimers, in which thefusion proteins of the multimer are alternatively aligned in oppositeorientations with respect to their N- and C-termini. Multimers (parallelor antiparallel) can be either homomultimers or heteromultimers. Thefusion protein is optionally produced in dimeric form; more preferably,the fusion is performed at the genetic level as described below, byjoining polynucleotide sequences corresponding to the two (or more)proteins, portions of proteins and/or peptides, such that a joined orfused protein is produced by a cell according to the joinedpolynucleotide sequence. A description of preparation for such fusionproteins is described with regard to U.S. Pat. No. 5,851,795 to Linsleyet al, which is hereby incorporated by reference as if fully set forthherein as a non-limiting example only.

Targeting Domains

The C1ORF32 polypeptides and fusion proteins can contain a targetingdomain to target the molecule to specific sites in the body. Optionaltargeting domains target the molecule to areas of inflammation.Exemplary targeting domains are antibodies, or antigen binding fragmentsthereof that are specific for inflamed tissue or to a proinflammatorycytokine including but not limited to IL17, IL-4, IL-6, IL-12, IL-21,IL-22, and IL-23. In the case of neurological disorders such as MultipleSclerosis, the targeting domain may target the molecule to the CNS ormay bind to VCAM-I on the vascular epithelium. Additional targetingdomains can be peptide aptamers specific for a proinflammatory molecule.In other embodiments, the C1ORF32 fusion protein can include a bindingpartner specific for a polypeptide displayed on the surface of an immunecell, for example a T cell. In still other embodiments, the targetingdomain specifically targets activated immune cells. Optional immunecells that are targeted include Th0, Th1, Th 17, Th2 and Th22 T cells,other cells that secrete, or cause other cells to secrete inflammatorymolecules including, but not limited to, IL-1beta, TNF-alpha, TGF-beta,IFN-gamma, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs, and Tregs. Forexample, a targeting domain for Tregs may bind specifically to CD25.

The above changes are intended as illustrations only of optional changesand are not meant to be limiting in any way. Furthermore, the aboveexplanation is provided for descriptive purposes only, without wishingto be bound by a single hypothesis.

Dimerization or multimierization can occur between or among two or morefusion proteins through dimerization or multimerization domains.Alternatively, dimerization or multimerization of fusion proteins canoccur by chemical crosslinking. The dimers or multimers that are formedcan be homodimeric/homomultimeric or heterodimeric/heteromultimeric. Thesecond polypeptide “partner” in the C1ORF32 fusion polypeptides may becomprised of one or more other proteins, protein fragments or peptidesas described herein, including but not limited to any immunoglobulin(Ig) protein or portion thereof, preferably the Fc region, or a portionof a biologically or chemically active protein such as thepapillomavirus E7 gene product, melanoma-associated antigen p97), andHIV env protein (gp120). The “partner” is optionally selected to providea soluble dimer/multimer and/or for one or more other biologicalactivities as described herein.

Addition of Groups

If a protein according to the present invention is a linear molecule, itis possible to place various functional groups at various points on thelinear molecule which are susceptible to or suitable for chemicalmodification. Functional groups can be added to the termini of linearforms of the protein according to at least some embodiments of theinvention. In some embodiments, the functional groups improve theactivity of the protein with regard to one or more characteristics,including but not limited to, improvement in stability, penetration(through cellular membranes and/or tissue barriers), tissuelocalization, efficacy, decreased clearance, decreased toxicity,improved selectivity, improved resistance to expulsion by cellularpumps, and the like. For convenience sake and without wishing to belimiting, the free N-terminus of one of the sequences contained in thecompositions according to at least some embodiments of the inventionwill be termed as the N-terminus of the composition, and the freeC-terminal of the sequence will be considered as the C-terminus of thecomposition. Either the C-terminus or the N-terminus of the sequences,or both, can be linked to a carboxylic acid functional groups or anamine functional group, respectively.

Non-limiting examples of suitable functional groups are described inGreen and Wuts, “Protecting Groups in Organic Synthesis”, John Wiley andSons, Chapters 5 and 7, 1991, the teachings of which are incorporatedherein by reference. Preferred protecting groups are those thatfacilitate transport of the active ingredient attached thereto into acell, for example, by reducing the hydrophilicity and increasing thelipophilicity of the active ingredient, these being an example for “amoiety for transport across cellular membranes”.

These moieties can optionally and preferably be cleaved in vivo, eitherby hydrolysis or enzymatically, inside the cell. (Ditter et al., J.Pharm. Sci. 57:783 (1968); Ditter et al., J. Pharm. Sci. 57:828 (1968);Ditter et al., J. Pharm. Sci. 58:557 (1969); King et al., Biochemistry26:2294 (1987); Lindberg et al., Drug Metabolism and Disposition 17:311(1989); and Tunek et al., Biochem. Pharm. 37:3867 (1988), Anderson etal., Arch. Biochem. Biophys. 239:538 (1985) and Singhal et al., FASEB J.1:220 (1987)). Hydroxyl protecting groups include esters, carbonates andcarbamate protecting groups Amine protecting groups include alkoxy andaryloxy carbonyl groups, as described above for N-terminal protectinggroups. Carboxylic acid protecting groups include aliphatic, benzylicand aryl esters, as described above for C-terminal protecting groups. Inone embodiment, the carboxylic acid group in the side chain of one ormore glutamic acid or aspartic acid residue in a composition of thepresent invention is protected, preferably with a methyl, ethyl, benzylor substituted benzyl ester, more preferably as a benzyl ester.

Non-limiting, illustrative examples of N-terminal protecting groupsinclude acyl groups (—CO—R1) and alkoxy carbonyl or aryloxy carbonylgroups (—CO—O—R1), wherein R1 is an aliphatic, substituted aliphatic,benzyl, substituted benzyl, aromatic or a substituted aromatic group.Specific examples of acyl groups include but are not limited to acetyl,(ethyl)-CO—, n-propyl-CO—, iso-propyl-CO—, n-butyl-CO—, sec-butyl-CO—,t-butyl-CO—, hexyl, lauroyl, palmitoyl, myristoyl, stearyl, oleoylphenyl-CO—, substituted phenyl-CO—, benzyl-CO— and (substitutedbenzyl)-CO—. Examples of alkoxy carbonyl and aryloxy carbonyl groupsinclude CH3-O—CO—, (ethyl)-O—CO—, n-propyl-O—CO—, iso-propyl-O—CO—,n-butyl-O—CO—, sec-butyl-O—CO—, t-butyl-O—CO—, phenyl-O— CO—,substituted phenyl-O—CO— and benzyl-O—CO—, (substituted benzyl)-O—CO—,Adamantan, naphtalen, myristoleyl, toluen, biphenyl, cinnamoyl,nitrobenzoy, toluoyl, furoyl, benzoyl, cyclohexane, norbornane, orZ-caproic. In order to facilitate the N-acylation, one to four glycineresidues can be present in the N-terminus of the molecule.

The carboxyl group at the C-terminus of the compound can be protected,for example, by a group including but not limited to an amide (i.e., thehydroxyl group at the C-terminus is replaced with —NH₂, —NHR₂ and—NR₂R₃) or ester (i.e. the hydroxyl group at the C-terminus is replacedwith —OR₂). R₂ and R₃ are optionally independently an aliphatic,substituted aliphatic, benzyl, substituted benzyl, aryl or a substitutedaryl group. In addition, taken together with the nitrogen atom, R₂ andR₃ can optionally form a C4 to C8 heterocyclic ring with from about 0-2additional heteroatoms such as nitrogen, oxygen or sulfur. Non-limitingsuitable examples of suitable heterocyclic rings include piperidinyl,pyrrolidinyl, morpholino, thiomorpholino or piperazinyl. Examples ofC-terminal protecting groups include but are not limited to —NH₂,—NHCH₃, —N(CH₃)₂, —NH(ethyl), —N(ethyl)₂, —N(methyl) (ethyl),—NH(benzyl), —N(C1-C4 alkyl)(benzyl), —NH(phenyl), —N(C1-C4 alkyl)(phenyl), —OCH₃, —O-(ethyl), —O-(n-propyl), —O-(n-butyl),—O-(iso-propyl), —O-(sec-butyl), —O-(t-butyl), —O-benz yl and —O-phenyl.

Substitution by Peptidomimetic Moieties

A “peptidomimetic organic moiety” can optionally be substituted foramino acid residues in the composition of this invention both asconservative and as non-conservative substitutions. These moieties arealso termed “non-natural amino acids” and may optionally replace aminoacid residues, amino acids or act as spacer groups within the peptidesin lieu of deleted amino acids. The peptidomimetic organic moietiesoptionally and preferably have steric, electronic or configurationalproperties similar to the replaced amino acid and such peptidomimeticsare used to replace amino acids in the essential positions, and areconsidered conservative substitutions. However such similarities are notnecessarily required. According to preferred embodiments of the presentinvention, one or more peptidomimetics are selected such that thecomposition at least substantially retains its physiological activity ascompared to the native protein according to the present invention.

Peptidomimetics may optionally be used to inhibit degradation of thepeptides by enzymatic or other degradative processes. Thepeptidomimetics can optionally and preferably be produced by organicsynthetic techniques. Non-limiting examples of suitable peptidomimeticsinclude D amino acids of the corresponding L amino acids, tetrazol(Zabrocki et al., J. Am. Chem. Soc. 110:5875-5880 (1988)); isosteres ofamide bonds (Jones et al., Tetrahedron Lett. 29: 3853-3856 (1988));LL-3-amino-2-propenidone-6-carboxylic acid (LL-Acp) (Kemp et al., J.Org. Chem. 50:5834-5838 (1985)). Similar analogs are shown in Kemp etal., Tetrahedron Lett. 29:5081-5082 (1988) as well as Kemp et al.,Tetrahedron Lett. 29:5057-5060 (1988), Kemp et al., Tetrahedron Lett.29:4935-4938 (1988) and Kemp et al., J. Org. Chem. 54:109-115 (1987).Other suitable but exemplary peptidomimetics are shown in Nagai andSato, Tetrahedron Lett. 26:647-650 (1985); Di Maio et al., J. Chem. Soc.Perkin Trans., 1687 (1985); Kahn et al., Tetrahedron Lett. 30:2317(1989); Olson et al., J. Am. Chem. Soc. 112:323-333 (1990); Garvey etal., J. Org. Chem. 56:436 (1990). Further suitable exemplarypeptidomimetics includehydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al., J.Takeda Res. Labs 43:53-76 (1989));1,2,3,4-tetrahydro-isoquinoline-3-carboxylate (Kazmierski et al., J. Am.Chem. Soc. 133:2275-2283 (1991)); histidine isoquinolone carboxylic acid(HIC) (Zechel et al., Int. J. Pep. Protein Res. 43 (1991)); (2S,3S)-methyl-phenylalanine, (2S, 3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and (2R, 3R)-methyl-phenylalanine (Kazmierskiand Hruby, Tetrahedron Lett. (1991)).

Exemplary, illustrative but non-limiting non-natural amino acids includebeta-amino acids (beta3 and beta2), homo-amino acids, cyclic aminoacids, aromatic amino acids, Pro and Pyr derivatives, 3-substitutedAlanine derivatives, Glycine derivatives, ring-substituted Phe and TyrDerivatives, linear core amino acids or diamino acids. They areavailable from a variety of suppliers, such as Sigma-Aldrich (USA) forexample.

Protein Chemical Modifications

In the present invention any part of a protein according to at leastsome embodiments of the invention may optionally be chemically modified,i.e. changed by addition of functional groups. For example the sideamino acid residues appearing in the native sequence may optionally bemodified, although as described below alternatively other parts of theprotein may optionally be modified, in addition to or in place of theside amino acid residues. The modification may optionally be performedduring synthesis of the molecule if a chemical synthetic process isfollowed, for example by adding a chemically modified amino acid.However, chemical modification of an amino acid when it is alreadypresent in the molecule (“in situ” modification) is also possible.

The amino acid of any of the sequence regions of the molecule canoptionally be modified according to any one of the following exemplarytypes of modification (in the peptide conceptually viewed as “chemicallymodified”). Non-limiting exemplary types of modification includecarboxymethylation, acylation, phosphorylation, glycosylation or fattyacylation. Ether bonds can optionally be used to join the serine orthreonine hydroxyl to the hydroxyl of a sugar. Amide bonds canoptionally be used to join the glutamate or aspartate carboxyl groups toan amino group on a sugar (Garg and Jeanloz, Advances in CarbohydrateChemistry and Biochemistry, Vol. 43, Academic Press (1985); Kunz, Ang.Chem. Int. Ed. English 26:294-308 (1987)). Acetal and ketal bonds canalso optionally be formed between amino acids and carbohydrates. Fattyacid acyl derivatives can optionally be made, for example, by acylationof a free amino group (e.g., lysine) (Toth et al., Peptides: Chemistry,Structure and Biology, Rivier and Marshal, eds., ESCOM Publ., Leiden,1078-1079 (1990)).

As used herein the term “chemical modification”, when referring to aprotein or peptide according to the present invention, refers to aprotein or peptide where at least one of its amino acid residues ismodified either by natural processes, such as processing or otherpost-translational modifications, or by chemical modification techniqueswhich are well known in the art. Examples of the numerous knownmodifications typically include, but are not limited to: acetylation,acylation, amidation, ADP-ribosylation, glycosylation, GPI anchorformation, covalent attachment of a lipid or lipid derivative,methylation, myristylation, pegylation, prenylation, phosphorylation,ubiquitination, or any similar process.

Other types of modifications optionally include the addition of acycloalkane moiety to a biological molecule, such as a protein, asdescribed in PCT Application No. WO 2006/050262, hereby incorporated byreference as if fully set forth herein. These moieties are designed foruse with biomolecules and may optionally be used to impart variousproperties to proteins.

Furthermore, optionally any point on a protein may be modified. Forexample, pegylation of a glycosylation moiety on a protein mayoptionally be performed, as described in PCT Application No. WO2006/050247, hereby incorporated by reference as if fully set forthherein. One or more polyethylene glycol (PEG) groups may optionally beadded to O-linked and/or N-linked glycosylation. The PEG group mayoptionally be branched or linear. Optionally any type of water-solublepolymer may be attached to a glycosylation site on a protein through aglycosyl linker.

Altered Glycosylation Proteins according to at least some embodiments ofthe invention may be modified to have an altered glycosylation pattern(i.e., altered from the original or native glycosylation pattern). Asused herein, “altered” means having one or more carbohydrate moietiesdeleted, and/or having at least one glycosylation site added to theoriginal protein.

Glycosylation of proteins is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequences,asparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to proteins according to at least someembodiments of the invention is conveniently accomplished by alteringthe amino acid sequence of the protein such that it contains one or moreof the above-described tripeptide sequences (for N-linked glycosylationsites). The alteration may also be made by the addition of, orsubstitution by, one or more serine or threonine residues in thesequence of the original protein (for O-linked glycosylation sites). Theprotein's amino acid sequence may also be altered by introducing changesat the DNA level.

Another means of increasing the number of carbohydrate moieties onproteins is by chemical or enzymatic coupling of glycosides to the aminoacid residues of the protein. Depending on the coupling mode used, thesugars may be attached to (a) arginine and histidine, (b) free carboxylgroups, (c) free sulfhydryl groups such as those of cysteine, (d) freehydroxyl groups such as those of serine, threonine, or hydroxyproline,(e) aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330, and in Aplin and Wriston, CRC Crit. Rev.Biochem., 22: 259-306 (1981).

Removal of any carbohydrate moieties present on proteins according to atleast some embodiments of the invention may be accomplished chemicallyor enzymatically. Chemical deglycosylation requires exposure of theprotein to trifluoromethanesulfonic acid, or an equivalent compound.This treatment results in the cleavage of most or all sugars except thelinking sugar (N-acetylglucosamine or N-acetylgalactosamine), leavingthe amino acid sequence intact.

Chemical deglycosylation is described by Hakimuddin et al., Arch.Biochem. Biophys., 259: 52 (1987); and Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on proteins canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138: 350 (1987).

Methods of Treatment

As used herein “therapeutic agent” is any one of the C1ORF32 proteinsand polypeptides fragments according to at least some embodiments of thepresent invention, and/or fusion proteins and/or multimeric proteinscomprising same, and/or nucleic acid sequence or fragments thereofencoding same.

As mentioned herein above, the therapeutic agents can be used to treatimmune related disorders as recited herein, and/or autoimmune disordersas recited herein, and/or infectious disorders as recited herein, and/orfor blocking and/or promoting immune costimulation mediated by theC1ORF32 polypeptides in a subject.

According to an additional aspect of the present invention thetherapeutic agents can be used to prevent pathologic inhibition of Tcell activity, such as that directed against chronic infections; and/orprevent pathologic stimulation of T cell activity, such as that directedagainst autoantigens in autoimmune diseases. For example, thesemolecules can be administered to cells in culture, in vitro or ex vivo,or to human subjects, e.g., in vivo, to treat, prevent and to diagnose avariety of disorders. Preferred subjects include human patients, havingdisorders mediated by cells expressing the C1ORF32 protein, and cellsthat possess C1ORF32 activity.

According to an additional aspect of the present invention thetherapeutic agents can be used to inhibit T cell activation, as can bemanifested for example by T cell proliferation and cytokine secretion.

Thus, according to an additional aspect of the present invention thereis provided a method of treating immune related disorders as recitedherein, and/or autoimmune disorders as recited herein, and/or infectiousdisorders as recited herein, and/or for blocking or promoting immunestimulation mediated by the C1ORF32 polypeptide in a subject byadministering to a subject in need thereof an effective amount of anyone of the therapeutic agents and/or a pharmaceutical compositioncomprising any of the therapeutic agents and further comprising apharmaceutically acceptable diluent or carrier.

The subject according to the present invention is a mammal, preferably ahuman which is diagnosed with one of the disease, disorder or conditionsdescribed hereinabove, or alternatively is predisposed to at least oneinfectious disorder, and/or immune related disorder.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented. Hence, the mammal to be treated herein may have beendiagnosed as having the disorder or may be predisposed or susceptible tothe disorder. “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.Preferably, the mammal is human.

As used herein the term “treating” refers to preventing, delaying theonset of, curing, reversing, attenuating, alleviating, minimizing,suppressing or halting the deleterious effects of the above-describeddiseases, disorders or conditions. It also includes managing the diseaseas described above. By “manage” it is meant reducing the severity of thedisease, reducing the frequency of episodes of the disease, reducing theduration of such episodes, reducing the severity of such episodes andthe like.

Treating, according to the present invention, can be effected byspecifically upregulating the amount and/or the expression of at leastone of the polypeptides of the present invention in the subject.

Optionally, upregulation may be effected by administering to the subjectat least one of the polypeptides of the present invention (e.g.,recombinant or synthetic) or an active portion thereof, as describedherein. However, since the bioavailability of large polypeptides maypotentially be relatively small due to high degradation rate and lowpenetration rate, administration of polypeptides is preferably confinedto small peptide fragments (e.g., about 100 amino acids). Thepolypeptide or peptide may optionally be administered in as part of apharmaceutical composition, described in more detail below.

It will be appreciated that treatment of the above-described diseasesaccording to at least some embodiments of the present invention may becombined with other treatment methods known in the art (i.e.,combination therapy), as described herein.

Immune System Related Disease Treatment

The therapeutic agents and/or a pharmaceutical composition comprisingsame, as recited herein, according to at least some embodiments of thepresent invention can also be used in combination with one or more ofthe following agents to regulate an immune response: soluble gp39 (alsoknown as CD40 ligand (CD40L), CD154, T-BAM, TRAP), soluble CD29, solubleCD40, soluble CD80 (e.g. ATCC 68627), soluble CD86, soluble CD28 (e.g.68628), soluble CD56, soluble Thy-1, soluble CD3, soluble TCR, solubleVLA-4, soluble VCAM-1, soluble LECAM-1, soluble ELAM-1, soluble CD44,antibodies reactive with gp39 (e.g. ATCC HB-10916, ATCC HB-12055 andATCC HB-12056), antibodies reactive with CD40 (e.g. ATCC HB-9110),antibodies reactive with B7 (e.g. ATCC HB-253, ATCC CRL-2223, ATCCCRL-2226, ATCC HB-301, ATCC HB-11341, etc), antibodies reactive withCD28 (e.g. ATCC HB-11944 or mAb 9.3), antibodies reactive with LFA-1(e.g. ATCC HB-9579 and ATCC TIB-213), antibodies reactive with LFA-2,antibodies reactive with IL-2, antibodies reactive with IL-12,antibodies reactive with IFN-gamma, antibodies reactive with CD2,antibodies reactive with CD48, antibodies reactive with any ICAM (e.g.,ICAM-1 (ATCC CRL-2252), ICAM-2 and ICAM-3), antibodies reactive withCTLA4 (e.g. ATCC HB-304), antibodies reactive with Thy-1, antibodiesreactive with CD56, antibodies reactive with CD3, antibodies reactivewith CD29, antibodies reactive with TCR, antibodies reactive with VLA-4,antibodies reactive with VCAM-1, antibodies reactive with LECAM-1,antibodies reactive with ELAM-1, antibodies reactive with CD44;L104EA29YIg, CD80 monoclonal antibodies (mAbs), CD86 mAbs, gp39 mAbs,CD40 mAbs, CD28 mAbs; anti-LFA1 mAbs, antibodies or other agentstargeting mechanisms of the immune system such as CD52 (alemtuzumab),CD25 (daclizumab), VLA-4 (natalizumab), CD20 (rituximab), IL2R(daclizumab) and MS4A1 (ocrelizumab); novel oral immunomodulating agentshave shown to prevent lymphocyte recirculation from lymphoid organs suchas fingolimod (FTY720) or leading to lymphocyte depletion such asmylinax (oral cladribine) or teriflunomide; and agents that preventimmunoactivation such as panaclar (dimethyl fumarate BG-12) orlaquinimod (ABR216062). Other combinations will be readily appreciatedand understood by persons skilled in the art. In some embodiments, thetherapeutic agents can be used to attenuate or reverse the activity of apro-inflammatory drug, and/or limit the adverse effects of such drugs.

As persons skilled in the art will readily understand, the combinationcan include the therapeutic agents and/or a pharmaceutical compositioncomprising same, according to at least some embodiments of the inventionand one other immunosuppressive agent; the therapeutic agents and/or apharmaceutical composition comprising same, as recited herein, with twoother immunosuppressive agents, the therapeutic agents and/or apharmaceutical composition comprising same, as recited herein, withthree other immunosuppressive agents, etc. The determination of theoptimal combination and dosages can be determined and optimized usingmethods well known in the art.

The therapeutic agent according to the present invention and one or moreother therapeutic agents can be administered in either order orsimultaneously.

The invention also encompasses the use of the therapeutic agents and/ora pharmaceutical composition comprising same according to at least someembodiments of the invention in combination with other pharmaceuticalagents to treat immune system diseases. For example, autoimmune diseasemay be treated with molecules according to at least some embodiments ofthe invention in conjunction with, immunosuppressants such ascorticosteroids, cyclosporin, cyclophosphamide, prednisone,azathioprine, methotrexate, rapamycin, tacrolimus, biological agentssuch as TNF-alpha blockers or antagonists, immunosuppressive agents(e.g., antibodies against other lymphocyte surface markers (e.g., CD40,alpha-4 integrin) or against cytokines), other fusion proteins (e.g.,CTLA-4-Ig (Orencia®), TNFR-Ig (Enbrel®)), TNF-alpha blockers such asEnbrel, Remicade, Cimzia and Humira, cyclophosphamide (CTX) (i.e.Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune™), methotrexate (MTX)(i.e. Rheumatrex®, Trexall®), belimumab (i.e. Benlysta®), or otherimmunosuppressive drugs (e.g., cyclosporin A, FK506-like compounds,rapamycin compounds, or steroids), anti-proliferatives, cytotoxicagents, or other compounds that may assist in immunosuppression. or anyother biological agent targeting any inflammatory cytokine, nonsteroidalantiinflammatory drugs/Cox-2 inhibitors, hydroxychloroquine,sulphasalazopryine, gold salts, etanercept, infliximab, mycophenolatemofetil, basiliximab, atacicept, rituximab, cytoxan, interferon beta-1a,interferon beta-1b, glatiramer acetate, mitoxantrone hydrochloride,anakinra and/or other biologics and/or intravenous immunoglobulin(IVIG). Non-limiting examples of such known therapeutics includeinterferons, such as IFN-beta-1a (REBIF®. AVONEX® and CINNOVEX®) andIFN-beta-1b (BETASERON®, EXTAVIA®, BETAFERON®, ZIFERON®); glatirameracetate (COPAXONE®), a polypeptide; natalizumab (TYSABRI®); andmitoxantrone (NOVANTRONE®), a cytotoxic agent.

Thus, treatment of multiple sclerosis using the agents according to atleast some embodiments of the present invention may be combined with,for example, any known therapeutic agent or method for treating multiplesclerosis. Non-limiting examples of such known therapeutic agent ormethod for treating multiple sclerosis include interferon class,IFN-beta-1a (REBIF®. AVONEX® and CINNOVEX®) and IFN-beta-1b (BETASERON®,EXTAVIA®, BETAFERON®, ZIFERON®); glatiramer acetate (COPAXONE®), apolypeptide; natalizumab (TYSABRI®); and mitoxantrone (NOVANTRONE®), acytotoxic agent, Fampridine (AMPYRA®). Other drugs includecorticosteroids, methotrexate, cyclophosphamide, azathioprine, andintravenous immunoglobulin (IVIG), inosine, Ocrelizumab (R1594), Mylinax(Caldribine), alemtuzumab (Campath), daclizumab (Zenapax),Panaclar/dimethyl fumarate (BG-12), Teriflunomide (HMR1726), fingolimod(FTY720), laquinimod (ABR216062), as well as Haematopoietic stem celltransplantation, Neurovax, Rituximab (Rituxan) BCG vaccine, low dosenaltrexone, helminthic therapy, angioplasty, venous stents, andalternative therapy, such as vitamin D, polyunsaturated fats, medicalmarijuana.

Thus, treatment of rheumatoid arthritis, using the agents according toat least some embodiments of the present invention may be combined with,for example, any known therapeutic agent or method for treatingrheumatoid arthritis. Non-limiting examples of such known therapeuticagents or methods for treating rheumatoid arthritis includeglucocorticoids, nonsteroidal anti-inflammatory drug (NSAID) such assalicylates, or cyclooxygenase-2 inhibitors, ibuprofen and naproxen,diclofenac, indomethacin, etodolac Disease-modifying antirheumatic drugs(DMARDs)-Oral DMARDs: Auranofin (Ridaura), Azathioprine (Imuran),Cyclosporine (Sandimmune, Gengraf, Neoral, generic), D-Penicillamine(Cuprimine), Hydroxychloroquine (Plaquenil), IM gold Gold sodiumthiomalate (Myochrysine) Aurothioglucose (Solganal), Leflunomide(Arava), Methotrexate (Rheumatrex), Minocycline (Minocin),Staphylococcal protein A immunoadsorption (Prosorba column),Sulfasalazine (Azulfidine). Biologic DMARDs: TNF-α blockers includingAdalimumab (Humira), Etanercept (Enbrel), Infliximab (Remicade),golimumab (Simponi), certolizumab pegol (Cimzia), and other BiologicalDMARDs, such as Anakinra (Kineret), Rituximab (Rituxan), Tocilizumab(Actemra), CD28 inhibitor including Abatacept (Orencia) and Belatacept.

Thus, treatment of IBD, using the agents according to at least someembodiments of the present invention may be combined with, for example,any known therapeutic agent or method for treating IBD. Non-limitingexamples of such known therapeutic agents or methods for treating IBDinclude immunosuppression to control the symptom, such as prednisone,Mesalazine (including Asacol, Pentasa, Lialda, Aspiro), azathioprine(Imuran), methotrexate, or 6-mercaptopurine, steroids, Ondansetron,TNF-α blockers (including infliximab, adalimumab golimumab, certolizumabpegol), Orencia (abatacept), ustekinumab (Stelara®), Briakinumab(ABT-874), Certolizumab pegol (Cimzia®), ITF2357 (givinostat),Natalizumab (Tysabri), Firategrast (SB-683699), Remicade (infliximab),vedolizumab (MLN0002), other drugs including GSK1605786 CCX282-B(Traficet-EN), AJM300, Stelara (ustekinumab), Semapimod (CNI-1493)tasocitinib (CP-690550), LMW Heparin MMX, Budesonide MMX, Simponi(golimumab), MultiStem®, Gardasil HPV vaccine, Epaxal Berna (virosomalhepatitis A vaccine), surgery, such as bowel resection, strictureplastyor a temporary or permanent colostomy or ileostomy; antifungal drugssuch as nystatin (a broad spectrum gut antifungal) and eitheritraconazole (Sporanox) or fluconazole (Diflucan); alternative medicine,prebiotics and probiotics, cannabis, Helminthic therapy or ova of theTrichuris suis helminth.

Thus, treatment of psoriasis, using the agents according to at leastsome embodiments of the present invention may be combined with, forexample, any known therapeutic agent or method for treating psoriasis.Non-limiting examples of such known therapeutics for treating psoriasisinclude topical agents, typically used for mild disease, phototherapyfor moderate disease, and systemic agents for severe disease.Non-limiting examples of topical agents: bath solutions andmoisturizers, mineral oil, and petroleum jelly; ointment and creamscontaining coal tar, dithranol (anthralin), corticosteroids likedesoximetasone (Topicort), Betamethasone, fluocinonide, vitamin D3analogues (for example, calcipotriol), and retinoids. Non-limitingexamples of phototherapy: sunlight; wavelengths of 311-313 nm, psoralenand ultraviolet A phototherapy (PUVA). Non-limiting examples of systemicagents: Biologics, such as interleukin antagonists, TNF-α blockersincluding antibodies such as infliximab (Remicade), adalimumab (Humira),golimumab, certolizumab pegol, and recombinant TNF-α decoy receptor,etanercept (Enbrel); drugs that target T cells, such as efalizumab(Xannelim/Raptiva), alefacept (Ameviv), dendritic cells such Efalizumab;monoclonal antibodies (MAbs) targeting cytokines, includinganti-IL-12/IL-23 (ustekinumab (brand name Stelara)) andanti-Interleukin-17; Briakinumab (ABT-874); small molecules, includingbut not limited to ISA247; Immunosuppressants, such as methotrexate,cyclosporine; vitamin A and retinoids (synthetic forms of vitamin A);and alternative therapy, such as changes in diet and lifestyle, fastingperiods, low energy diets and vegetarian diets, diets supplemented withfish oil rich in Vitamin A and Vitamin D (such as cod liver oil), Fishoils rich in the two omega-3 fatty acids eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA) and contain Vitamin E Ichthyotherapy,Hypnotherapy, cannabis.

Thus, treatment of type 1 diabetes, using the agents according to atleast some embodiments of the present invention may be combined with,for example, any known therapeutic agent or method for treating typeldiabetes. Non-limiting examples of such known therapeutics for treatingtype 1 diabetes include insulin, insulin analogs, islet transplantation,stem cell therapy including PROCHYMAL®, non-insulin therapies such asit-1beta inhibitors including Anakinra (Kineret®), Abatacept (Orencia®),Diamyd, alefacept (Ameviv®), Otelixizumab, DiaPep277 (Hsp60 derivedpeptide), Alpha 1-Antitrypsin, Prednisone, azathioprine, Ciclosporin,E1-INT (an injectable islet neogenesis therapy comprising an epidermalgrowth factor analog and a gastrin analog), statins including Zocor®,Simlup®, Simcard®, Simvacor®, Sitagliptin (dipeptidyl peptidase (DPP-4)inhibitor), Anti-CD3 mAb (e.g., Teplizumab); CTLA4-Ig (abatacept), AntiIL-1Beta (Canakinumab), Anti-CD20 mAb (e.g, rituximab).

Thus, treatment of uveitis, using the agents according to at least someembodiments of the present invention may be combined with, for example,any known therapeutic agent or method for treating uveitis. Non-limitingexamples of such known therapeutics for treating uveitis includecorticosteroids, topical cycloplegics, such as atropine or homatropine,or injection of PSTTA (posterior subtenon triamcinolone acetate),antimetabolite medications, such as methotrexate, TNF-α blockers(including infliximab, adalimumab, etanercept, golimumab, certolizumabpegol).

Thus, treatment for Sjogren's syndrome, using the agents according to atleast some embodiments of the present invention may be combined with,for example, any known therapeutic agent or method for treating forSjogren's syndrome. Non-limiting examples of such known therapeutics fortreating for Sjogren's syndrome include Cyclosporine, pilocarpine(Salagen) and cevimeline (Evoxac), Hydroxychloroquine (Plaquenil),cortisone (prednisone and others) and/or azathioprine (Imuran) orcyclophosphamide (Cytoxan), Dexamethasone, Thalidomide,Dehydroepiandrosterone, NGX267, Rebamipide, FID 114657, Etanercept,Raptiva, Belimumab, MabThera (rituximab); Anakinra, intravenous immuneglobulin (IVIG), Allogeneic Mesenchymal Stem Cells (AlloMSC), Automaticneuro-electrostimulation by “Saliwell Crown”.

Thus, treatment for systemic lupus erythematosus, using the agentsaccording to at least some embodiments of the present invention may becombined with, for example, any known therapeutic agent or method fortreating for systemic lupus erythematosus. Non-limiting examples of suchknown therapeutics for treating for systemic lupus erythematosus includecorticosteroids and Disease-modifying antirheumatic drugs (DMARDs),commonly anti-malarial drugs such as plaquenil and immunosuppressants(e.g. methotrexate and azathioprine) Hydroxychloroquine, cytotoxic drugs(e.g., cyclophosphamide and mycophenolate), Hydroxychloroquine (HCQ),Benlysta (belimumab), nonsteroidal anti-inflammatory drugs, Prednisone,Cellcept, Prograf, Atacicept, Lupuzor, Intravenous Immunoglobulins(IVIGs), CellCept (mycophenolate mofetil), Orencia, CTLA4-IgG4m(RG2077), rituximab, Ocrelizumab, Epratuzumab, CNTO 136, Sifalimumab(MEDI-545), A-623 (formerly AMG 623), AMG 557, Rontalizumab, paquinimod(ABR-215757), LY2127399, CEP-33457, Dehydroepiandrosterone,Levothyroxine, abetimus sodium (LIP 394), Memantine, Opiates, Rapamycin,Renal transplantation, stem cell transplantation.

The therapeutic agents and/or a pharmaceutical composition comprisingsame, as recited herein, according to at least some embodiments of theinvention, may be administered as the sole active ingredient or togetherwith other drugs in immunomodulating regimens or other anti-inflammatoryagents e.g. for the treatment or prevention of allo- or xenograft acuteor chronic rejection or inflammatory or autoimmune disorders, or toinduce tolerance.

For example, it may be used in combination with a calcineurin inhibitor,e.g. cyclosporin A or FK506; an immunosuppressive macrolide, e.g.rapamycine or a derivative thereof; e.g.40-0-(2-hydroxy)ethyl-rapamycin, a lymphocyte homing agent, e.g. FTY720or an analog thereof, corticosteroids; cyclophosphamide; azathioprene;methotrexate; leflunomide or an analog thereof; mizoribine; mycophenolicacid; mycophenolate mofetil; 15-deoxyspergualine or an analog thereof;immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies toleukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD 11a/CD18, CD7, CD25,CD 27, B7, CD40, CD45, CD58, CD 137, ICOS, CD150 (SLAM), OX40, 4-1BB ortheir ligands; or other immunomodulatory compounds, e.g. CTLA4-Ig(abatacept, ORENCIA® or belatacept), CD28-Ig, B7-H4-Ig, or othercostimulatory agents, or adhesion molecule inhibitors, e.g. mAbs or lowmolecular weight inhibitors including LFA-1 antagonists, Selectinantagonists and VLA-4 antagonists.

Where the therapeutic agents and/or a pharmaceutical compositioncomprising same, as recited herein, according to at least someembodiments of the invention are administered in conjunction with otherimmunosuppressive/immunomodulatory or anti-inflammatory therapy, e.g. asherein above specified, dosages of the co-administeredimmunosuppressant, immunomodulatory or anti-inflammatory compound willof course vary depending on the type of co-drug employed, e.g. whetherit is a steroid or a cyclosporin, on the specific drug employed, on thecondition being treated and so forth.

According to at least some embodiments of the present invention, thereis provided use of a combination of the therapeutic agents and/or apharmaceutical composition comprising same, as recited herein, and aknown therapeutic agent effective for treating infection.

The therapeutic agents and/or a pharmaceutical composition comprisingsame, as recited herein, can be administered in combination with one ormore additional therapeutic agents used for treatment of bacterialinfections, including, but not limited to, antibiotics includingAminoglycosides, Carbapenems, Cephalosporins, Macrolides, Lincosamides,Nitrofurans, penicillins, Polypeptides, Quinolones, Sulfonamides,Tetracyclines, drugs against mycobacteria including but not limited toClofazimine, Cycloserine, Cycloserine, Rifabutin, Rifapentine,Streptomycin and other antibacterial drugs such as Chloramphenicol,Fosfomycin, Metronidazole, Mupirocin, and Tinidazole.

The therapeutic agents and/or a pharmaceutical composition comprisingsame, as recited herein, can be administered in combination with one ormore additional therapeutic agents used for treatment of viralinfections, including, but not limited to, antiviral drugs such asoseltamivir (brand name Tamiflu) and zanamivir (brand name Relenza)Arbidol-adamantane derivatives (Amantadine, Rimantadine)-neuraminidaseinhibitors (Oseltamivir, Laninamivir, Peramivir, Zanamivir) nucleotideanalog reverse transcriptase inhibitor including Purine analogue guanine(Aciclovir#/Valacyclovir, Ganciclovir/Valganciclovir,Penciclovir/Famciclovir) and adenine (Vidarabine), Pyrimidine analogue,uridine (Idoxuridine, Trifluridine, Edoxudine), thymine (Brivudine),cytosine (Cytarabine); Foscarnet; Nucleoside analogues/NARTIs:Entecavir, Lamivudine, Telbivudine, Clevudine; Nucleotideanalogues/NtRTIs: Adefovir, Tenofovir; Nucleic acid inhibitors such asCidofovir; InterferonInterferon alfa-2b, Peginterferon alfa-2a;Ribavirin#/Taribavirin; antiretroviral drugs including zidovudine,lamivudine, abacavir, lopinavir, ritonavir, tenofovir/emtricitabine,efavirenz each of them alone or a various combinations, gp41(Enfuvirtide), Raltegravir, protease inhibitors such as Fosamprenavir,Lopinavir and Atazanavir, Methisazone, Docosanol, Fomivirsen,Tromantadine.

The therapeutic agents and/or a pharmaceutical composition comprisingsame, as recited herein, can be administered in combination with one ormore additional therapeutic agents used for treatment of fungalinfections, including, but not limited to, antifungal drugs of thePolyene antifungals, Imidazole, triazole, and thiazole antifungals,Allylamines, Echinocandins or other anti fungal drugs.

Alternatively or additionally, an upregulating method may optionally beeffected by specifically upregulating the amount (optionally expression)in the subject of at least one of the polypeptides of the presentinvention or active portions thereof.

As is mentioned hereinabove and in the Examples section which follows,the biomolecular sequences of this aspect of the present invention maybe used as valuable therapeutic tools in the treatment of diseases,disorders or conditions in which altered activity or expression of thewild-type gene product (known protein) is known to contribute todisease, disorder or condition onset or progression. For example, incase a disease is caused by overexpression of a membrane bound-receptor,a soluble variant thereof may be used as an antagonist which competeswith the receptor for binding the ligand, to thereby terminate signalingfrom the receptor.

According to at least some embodiments, immune cells, preferably Tcells, can be contacted in vivo or ex vivo with the therapeutic agentsto modulate immune responses. The T cells contacted with the therapeuticagents can be any cell which expresses the T cell receptor, includingα/β and γ/δ T cell receptors. T-cells include all cells which expressCD3, including T-cell subsets which also express CD4 and CDS. T-cellsinclude both naive and memory cells and effector cells such as CTL.T-cells also include cells such as Th1, Tc1, Th2, Tc2, Th3, Th17, Th22,Treg, and Tr1 cells. T-cells also include NKT-cells and similar uniqueclasses of the T-cell lineage.

In a further embodiment, the additional therapeutic agent functions toinhibit or reduce T cell activation through a separate pathway. In onesuch embodiment, the additional therapeutic agent is a CTL A-4 fusionprotein, such as CTLA-4-Ig (abatacept). CTLA-4-Ig fusion proteinscompete with the co-stimulatory receptor, CD28, on T cells for bindingto CD80/CD86 (B7-1/B7-2) on antigen presenting cells, and thus functionto inhibit T cell activation. In another embodiment, the additionaltherapeutic agent is a CTLA-4-Ig fusion protein known as belatacept.Belatacept contains two amino acid substitutions (L104E and A29Y) thatmarkedly increase its avidity to CD86 in vivo. In another embodiment,the additional therapeutic agent is Maxy-4.

In another embodiment, the second therapeutic agent is cyclophosphamide(CTX). Cyclophosphamide (the generic name for Endoxan®, Cytoxan®,Neosar®, Procytox®, Revimmune™), also known as cytophosphane, is anitrogen mustard alkylating agent from the oxazophorines group. It isused to treat various types of cancer and some autoimmune disorders. Ina further embodiment, IgC1ORF32 polypeptides, fragments or fusionproteins thereof and CTX are coadministered in effective amount toprevent or treat a chronic autoimmune disease or disorder such asSystemic lupus erythematosus (SLE). Cyclophosphamide (CTX) is theprimary drug used for diffuse proliferative glomerulonephritis inpatients with renal lupus. In some embodiments the combination therapyis administered in an effective amount to reduce the blood or serumlevels of anti-double stranded DNA (anti-ds DNA) auto antibodies and/orto reduce proteinuria in a patient in need thereof.

In another embodiment, the second therapeutic is Tysabri or anothertherapeutic for MS. In a further embodiment, IgC1ORF32 polypeptides,fragments or fusion proteins thereof is cycled with Tysabri or usedduring a drug holiday in order to allow less frequent dosing with thesecond therapeutic and reduce the risk of side effects such as PML andto prevent resistance to the second therapeutic.

In another embodiment, the second therapeutic agent preferentiallytreats chronic inflammation, whereby the treatment regimen targets bothacute and chronic inflammation. In a further embodiment the secondtherapeutic is a TNF-alpha blocker.

In another embodiment, the second therapeutic agent is a small moleculethat inhibits or reduces differentiation, proliferation, activity,and/or cytokine production and/or secretion by Th1, Th17, Th22, and/orother cells that secrete, or cause other cells to secrete, inflammatorymolecules, including, but not limited to, IL-1beta, TNF-alpha, TGF-beta,IFN-gamma, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs. In anotherembodiment, the second therapeutic agent is a small molecule thatinteracts with Tregs, enhances Treg activity, promotes or enhances IL-10secretion by Tregs, increases the number of Tregs, increases thesuppressive capacity of Tregs, or combinations thereof.

Typically useful small molecules are organic molecules, preferably smallorganic compounds having a molecular weight of more than 100 and lessthan about 2,500 daltons, more preferably between 100 and 2000, morepreferably between about 100 and about 1250, more preferably betweenabout 100 and about 1000, more preferably between about 100 and about750, more preferably between about 200 and about 500 daltons. Smallmolecules comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The smallmolecules often comprise cyclical carbon or heterocyclic structuresand/or aromatic or polyaromatic structures substituted with one or moreof the above functional groups. Small molecules also includebiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof. In one embodiment, the small molecule is retinoic acid or aderivative thereof. The examples below demonstrate that retinoic acidinhibits or reduces differentiation and/or activity of ThI 7 cells. In afurther embodiment, the compositions are used in combination orsuccession with compounds that increase Treg activity or production.Exemplary Treg enhancing agents include but are not limited toglucocorticoid fluticasone, salmeteroal, antibodies to IL-12, IFN-gamma,and IL-4; vitamin D3, and dexamethasone, and combinations thereof.Antibodies to other proinflammatory molecules can also be used incombination or alternation with the disclosed C1ORF32 polypeptideselected from the group consisting of SEQ ID NOs: 29, 30, 41-105, or afragment, variant, a homolog, a fusion protein or a conjugate thereof.Preferred antibodies bind to IL-6, IL-23, IL-22 or IL-21.

As used herein the term “rapamycin compound” includes the neutraltricyclic compound rapamycin, rapamycin derivatives, rapamycin analogs,and other macrolide compounds which are thought to have the samemechanism of action as rapamycin (e.g., inhibition of cytokinefunction). The language “rapamycin compounds” includes compounds withstructural similarity to rapamycin, e.g., compounds with a similarmacrocyclic structure, which have been modified to enhance theirtherapeutic effectiveness. Exemplary Rapamycin compounds are known inthe art. The language “FK506-Hke compounds” includes FK506, and FK506derivatives and analogs, e.g., compounds with structural similarity toFK506, e.g., compounds with a similar macrocyclic structure which havebeen modified to enhance their therapeutic effectiveness. Examples ofFK506-like compounds include, for example, those described in WO00101385. Preferably, the language “rapamycin compound” as used hereindoes not include FK506-like compounds.

Other suitable therapeutics include, but are not limited to,anti-inflammatory agents. The anti-inflammatory agent can benon-steroidal, steroidal, or a combination thereof. One embodimentprovides oral compositions containing about 1% (w/w) to about 5% (w/w),typically about 2.5 (w/w) or an anti-inflammatory agent. Representativeexamples of non-steroidal anti-inflammatory agents include, withoutlimitation, oxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam;salicylates, such as aspirin, disalcid, benorylate, trilisate, safapryn,solprin, diflunisal, and fendosal; acetic acid derivatives, such asdiclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac,furofenac, tiopinac, zidometacin, acematacin, fentiazac, zomepirac,clmdanac, oxepinac, felbmac, and ketorolac; fenamates, such asmefenamic, meclofenamic, flufenamic, niflumic, and tolfenamic acids;propionic acid derivatives, such as ibuprofen, naproxen, benoxaprofen,flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropfen, pirprofen,carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen,alminoprofen, and tiaprofenic; pyrazoles, such as phenylbutazone,oxyphenbutazone, feprazone, azapropazone, and trimethazone. Mixtures ofthese non-steroidal anti-inflammatory agents may also be employed.

Representative examples of steroidal anti-inflammatory drugs include,without limitation, corticosteroids such as hydrocortisone,hydroxyl-triamcinolone, alpha-methyl dexamethasone,dexamethasone-phosphate, beclomethasone dipropionates, clobetasolvalerate, desonide, desoxymethasone, desoxycorticosterone acetate,dexamethasone, dichlorisone, diflorasone diacetate, diflucortolonevalerate, fluadrenolone, fluclorolone acetonide, fludrocortisone,flumethasone pivalate, fiuosinolone acetonide, fluocinonide, flucortinebutylesters, fluocortolone, fluprednidene (fluprednylidene) acetate,flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisonebutyrate, methylprednisolone, triamcinolone acetonide, cortisone,cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate,fluradrenolone, fludrocortisone, diflurosone diacetate, fluradrenoloneacetonide, medrysone, amcinafel, amcinafide, betamethasone and thebalance of its esters, chloroprednisone, chlorprednisone acetate,clocortelone, clescinolone, dichlorisone, diflurprednate, flucloronide,flunisolide, fluoromethalone, fluperolone, fluprednisolone,hydrocortisone valerate, hydrocortisone cyclopentylpropionate,hydrocortamate, meprednisone, paramethasone, prednisolones prednisone,beclomethasone dipropionate, triamcinolone, and mixtures thereof.

Methods of Therapeutic Use

The C1ORF32 polypeptides, fragments or fusion proteins thereof areuseful as therapeutic agents. According to at least some embodiments,immune cells, preferably T cells, can be contacted in vivo or ex vivowith C1ORF32 fusion polypeptides to decrease or inhibit immune responsesincluding, but not limited to inflammation. According to at least someother embodiments, immune cells, preferably T cells, can be contacted invivo or ex vivo with C1ORF32 fusion polypeptides to decrease or inhibitT cell exhaustion, optionally in combination with another therapeuticagent. In both cases, preferably the costimulatory pathway is modulatedto achieve a desired immune system balance.

In either case, the T cells contacted with C1ORF32 fusion polypeptidescan be any cell which expresses the T cell receptor, including α/β andγ/δ T cell receptors. T-cells include all cells which express CD3,including T-cell subsets which also express CD4 and CDS. T-cells includeboth naive and memory cells and effector cells such as CTL. T-cells alsoinclude cells such as Th1, Tc1, Th2, Tc2, Th3, Th17, Th22, Treg, and Tr1cells. T-cells also include NKT-cells and similar unique classes of theT-cell lineage. For example the compositions can be used to modulateTh1, Th17, Th22, or other cells that secrete, or cause other cells tosecrete, inflammatory molecules, including, but not limited to,IL-1beta, TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6, IL-23, IL-22,IL-21, and MMPs. The compositions can also be used to increase orpromote the activity of Tregs, increase the production of cytokines suchas IL-10 from Tregs, increase the differentiation of Tregs, increase thenumber of Tregs, or increase the survival of Tregs. The compositions canalso be used to increase or promote the activity of Th2 cells, increasethe production of cytokines such as IL-10 or IL-4 from Th2 cells,increase the differentiation of Th2 cells, increase the number of Th2cells, or increase the survival of Th2 cells.

Although the below discussion applies generally to the reversal of Tcell exhaustion, preferably for treatment of such exhaustion memory Tcells are treated. Optionally, additionally or alternatively, treatmentof T cell exhaustion encompasses the modulation of cytokine secretion,or other cells that secrete, or cause other cells to secrete,inflammatory molecules, including, but not limited to, IL-2, TNF-alpha,IFN-gamma, 15 Granzyme B and MMPs or modulate the expression of molecuesof the costimulatory/coinhibtory-family including, but not limited toPD-1, Tim3, CTLA4 and LAGS.

In some embodiments, the disclosed C1ORF32 polypeptide, selected fromthe group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,variant, a homolog, a fusion protein or a conjugate thereof, areadministered in combination with a second therapeutic. Combinationtherapies may be useful in immune modulation. In some embodiments,C1ORF32 polypeptide, selected from the group consisting of SEQ ID NOs:29, 30, 41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof, can be used to attenuate or reverse the activity of apro-inflammatory drug, and/or limit the adverse effects of such drugs.

Other immune cells that can be treated with the disclosed C1ORF32polypeptides, fragments or fusion thereof include T cell precursors,antigen presenting cells such as dendritic cells and monocytes or theirprecursors, B cells or combinations thereof. The C1ORF32 compositionscan be used to modulate the production of antibodies by B cells bycontacting the B cells with an effective amount of the C1ORF32composition to inhibit or reduce antibody production by the B cellrelative to a control. The C1ORF32 compositions can also modulate theproduction of cytokines by the B cells.

Methods of Treating Inflammatory Responses

The C1ORF32 polypeptides, fragments or fusion proteins thereof, selectedfrom the group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,variant, a homolog, a fusion protein or a conjugate thereof, accordingto at least some embodiments of the present invention inhibit T cellactivation, as manifested by T cell proliferation and cytokinesecretion. Specifically, the proteins inhibit T Th1 and Th17 responses,while promoting Th2 responses.

The C1ORF32 polypeptides, fragments or fusion proteins thereof, selectedfrom the group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,variant, a homolog, a fusion protein or a conjugate thereof, accordingto at least some embodiments of the present invention are potentiallyused for therapy of diseases that require down-regulation ofcostimulatory pathways and or such that require downregulation of Th1and/or Th17 responses.

A further embodiment provides methods for treating or alleviating one ormore symptoms of inflammation. In a further embodiment, the compositionsand methods disclosed are useful for treating chronic and persistentinflammation. Inflammation in general can be treated using the disclosedC1ORF32 polypeptides or fragment or fusions thereof.

According to at least some embodiments of the present invention, thereis provided use of an isolated C1ORF32 polypeptide as described hereinor a fusion protein comprising an isolated C1ORF32 polypeptide asdescribed herein, optionally in a pharmaceutical composition comprisinga pharmaceutically acceptable diluent or carrier, for treatment of animmune related disorder and/or infection.

An immune response including inflammation can be inhibited or reduced ina subject, preferably a human, by administering an effective amount ofC1ORF32 polypeptide selected from the group consisting of SEQ ID NOs:29, 30, 41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof, to inhibit or reduce the biological activity of animmune cell or to reduce the amounts of proinflammatory molecules at asite of inflammation. Exemplary proinflammatory molecules include, butare not limited to, IL-1beta, TNF-alpha, TGF-beta, IFN-gamma, IL-17,IL-6, IL-23, IL-22, IL-21, and MMPs.

Th1 and Th17 are exemplary T cells that can be targeted for inhibitionby C1ORF32 polypeptide selected from the group consisting of SEQ ID NOs:29, 30, 41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof, to inhibit or reduce inflammation.

Without wishing to be limited by a single hypothesis for this biologicalmechanism or any other biological mechanism described herein, theC1ORF32 polypeptides, fragments or fusion proteins thereof are usefulfor treating inflammation by any or all of the following: inhibiting orreducing differentiation of Th1, Th17, Th22, and/or other cells thatsecrete, or cause other cells to secrete, inflammatory molecules,including, but not limited to, IL-1beta, TNF-alpha, TGF-beta, IFN-gamma,IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs; inhibiting or reducingactivity of ThI, Th 17, Th22, and/or other cells that secrete, or causeother cells to secrete, inflammatory molecules, including, but notlimited to, IL-1beta, TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6,IL-23, IL-22, IL-21, and MMPs; inhibiting or reducing the Th1 and/orTh17 pathways; inhibiting or reducing cytokine production and/orsecretion by Th1, Th17, Th22, and/or other cells that secrete, or causeother cells to secrete, inflammatory molecules, including, but notlimited to, IL-1beta, TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6 IL-23,IL-22, IL-21, and MMPs; inhibiting or reducing proliferation of Th1,Th17, Th22, and/or other cells that secrete, or cause other cells tosecrete, inflammatory molecules, including, but not limited to,IL-1beta, TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6, IL-23, IL-22,IL-21, and MMPs.

Additionally, C1ORF32 polypeptide selected from the group consisting ofSEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusionprotein or a conjugate thereof can also enhance Th2 immune responses.C1ORF32 polypeptide selected from the group consisting of SEQ ID NOs:29, 30, 41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof can also act directly on Th2 cells to promote orenhance production of IL-4, IL-5 or IL-10, or to increase the number ofTh2 cells, resulting in inhibition of Th1 and/or Th17, and in immunemodulation via a Th1/Th2 shift.

Additionally, C1ORF32 polypeptide selected from the group consisting ofSEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusionprotein or a conjugate thereof can cause Tregs to have an enhancedsuppressive effect on an immune response. Tregs can suppressdifferentiation, proliferation, activity, and/or cytokine productionand/or secretion by Th1, Th17, Th22, and/or other cells that secrete, orcause other cells to secrete, inflammatory molecules, including, but notlimited to, IL-1beta, TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6,IL-23, IL-22, IL-21, and MMPs. For example, C1ORF32 polypeptide selectedfrom the group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,variant, a homolog, a fusion protein or a conjugate thereof can causeTregs to have an enhanced suppressive effect on Th1 and/or Th17 cells toreduce the level of IFN-gamma and IL-17 produced, respectively.C1ORF32polypeptide selected from the group consisting of SEQ ID NOs: 29,30, 41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof can also act directly on Tregs to promote or enhanceproduction of IL-10 to suppress the Th1 and/or Th17 pathway, and/or toincrease the number of Tregs.

Additionally, C1ORF32 polypeptide selected from the group consisting ofSEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusionprotein or a conjugate thereof can cause Th2 to have an enhancedmodulatory effect on an immune response. Th2 cells can modulatedifferentiation, proliferation, activity, and/or cytokine productionand/or secretion by Th1, Th17, Th22, and/or other cells that secrete, orcause other cells to secrete, inflammatory molecules, including, but notlimited to, IL-1beta, TNF-alpha, TGF-beta, IFN-gamma, IL-17, IL-6,IL-23, IL-22, IL-21, and MMPs. For example, C1ORF32 polypeptide selectedfrom the group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,variant, a homolog, a fusion protein or a conjugate thereof can causeTh2 cells to have an enhanced modulatory effect on Th1 and/or Th17 cellsto reduce the level of IFN-gamma and IL-17 produced, respectively.C1ORF32 polypeptide selected from the group consisting of SEQ ID NOs:29, 30, 41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof can also act directly on Th2 cells to promote orenhance production of IL-10 to suppress the Th1 and/or Th17 pathway,and/or to increase the number of Th2 cells.

Without wishing to be limited by a single hypothesis, it is believedthat C1ORF32 polypeptide selected from the group consisting of SEQ IDNOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusion proteinor a conjugate thereof acts at multiple points in multiple T cellpathways. For example, polypeptide selected from the group consisting ofSEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusionprotein or a conjugate thereof can inhibit the differentiation of naiveT cells into either Th1 or Th17 cells. Alternatively, polypeptideselected from the group consisting of SEQ ID NOs: 29, 30, 41-105, or afragment, variant, a homolog, a fusion protein or a conjugate thereofcan interact with Th1 cells or Th17 cells, or both to inhibit or reducethe production of proinflammatory molecules.

Additionally, C1ORF32 polypeptide selected from the group consisting ofSEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusionprotein or a conjugate thereof may increase the differentiation ofand/or promote Th2 responses resulting in an immunomdulatory effect onthe Th1 and/or Th17 pathways to reduce the level of INF-gamma and/orIL-17 produced. C1ORF32 polypeptide selected from the group consistingof SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, afusion protein or a conjugate thereof enhances the production of IL-10from cells such as Th2 and/or Tregs, which in turn inhibits the activityof Th1 and/or Th17 cells.

Additionally, C1ORF32 polypeptide selected from the group consisting ofSEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusionprotein or a conjugate thereof can affect Tregs to have an enhancedsuppressive effect on Th1 and/or Th17 pathways to reduce the level ofINF-gamma and/or IL-17 produced. Additionally, C1ORF32 polypeptideselected from the group consisting of SEQ ID NOs: 29, 30, 41-105, or afragment, variant, a homolog, a fusion protein or a conjugate thereofcan enhance the production of IL-10 which inhibits the activity of Th1and/or Th17 cells.

Inhibition of Th1 Responses

a. Inhibition of Th1 Development

One method for inhibiting or reducing inflammation includesadministering an effective amount of a C1ORF32 polypeptide selected fromthe group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,variant, a homolog, a fusion protein or a conjugate thereof to inhibitTh1 development in a subject in need thereof. Inflammation can beinhibited or reduced by blocking naive T cells from differentiating intoTh1 cells by administering C1ORF32 polypeptide selected from the groupconsisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, ahomolog, a fusion protein or a conjugate thereof. In one embodiment, theC1ORF32 polypeptide selected from the group consisting of SEQ ID NOs:29, 30, 41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof may inhibit or reduce proliferation of Th1 cells.C1ORF32 polypeptide selected from the group consisting of SEQ ID NOs:29, 30, 41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof may also reduce naive T cells from differentiatinginto Th1 cells, by blocking antigen presenting cell maturation.Alternatively, C1ORF32 polypeptide selected from the group consisting ofSEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusionprotein or a conjugate thereof increase the differentiation of Th2 cellsand thereby reduce the number of Th1 cells in a subject. By restrictingthe number of Th1 cells that can develop in the subject, the amount ofproinflammatory molecules such as INF-gamma can be reduced or contained.INF-gamma stimulates the production or release of other proinflammatorymolecules including IL-1beta, TNF-alpha, and MMPs. Thus, by controllingthe number of Th1 cells in a subject, the levels of these otherproinflammatory molecules can be controlled, thereby reducinginflammatory responses.

b. Inhibition of Proinflammatory Molecules

Another embodiment provides a method of inhibiting or reducinginflammation in a subject by administering to the subject an effectiveamount of a C1ORF32 polypeptide selected from the group consisting ofSEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusionprotein or a conjugate thereof to inhibit or reduce production ofproinflammatory molecules by Th1 cells.

Exemplary proinflammatory molecules produced by Th1 cells includesIFN-gamma. In this embodiment the C1ORF32 polypeptide selected from thegroup consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant,a homolog, a fusion protein or a conjugate thereof can interact directlywith the Th1 cell and inhibit or reduce IFN-gamma production by the Th1cells. In this embodiment, the amount of proinflammatory molecules isregulated rather than the population of Th1 cells.

Inhibition of Th17 Responses a. Inhibition of Th17 Development

Inflammation can also be inhibited or reduced in a subject byadministering an effective amount of a C1ORF32 polypeptide selected fromthe group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,variant, a homolog, a fusion protein or a conjugate thereof, to inhibitor block naive T cells from developing into Th17 cells. In oneembodiment, the C1ORF32 polypeptide selected from the group consistingof SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, afusion protein or a conjugate thereof increases the suppressive activityof Tregs on the differentiation of naive T cells into Th17 cells by anamount sufficient to reduce the number of Th17 cells in a subject.Alternatively, the C1ORF32 polypeptide selected from the groupconsisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, ahomolog, a fusion protein or a conjugate thereof inhibits or reducesproliferation of Th17 cells. C1ORF32 polypeptide selected from the groupconsisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, ahomolog, a fusion protein or a conjugate thereof may also reduce naive Tcells from differentiating into Th17 cells, by blocking antigenpresenting cell maturation. By reducing the population of Th17 cells ina subject, the amount of IL-17 can be reduced, as well as IL-22 andIL-21. IL-17 is a proinflammatory cytokine that causes increases inother proinflammatory molecules such as IL-1beta, TNF-alpha, and MMPs.Thus, by reducing the amount of IL-17 these other proinflammatorymolecules can be reduced, thereby reducing or inhibiting inflammation.

b. Inhibition of IL-17 Production

Still another embodiment provides a method for treating inflammation ina subject by administering an effective amount of C1ORF32 polypeptideselected from the group consisting of SEQ ID NOs: 29, 30, 41-105, or afragment, variant, a homolog, a fusion protein or a conjugate thereof,to inhibit production of IL-17 by Th17 cells, as well as IL-22 andIL-21. In this embodiment, the C1ORF32 polypeptide selected from thegroup consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant,a homolog, a fusion protein or a conjugate thereof can act directly onTh17 cells, for example by binding to Th17 cells resulting in inhibitionof IL-17 (or IL-22 and IL-21) production by those Th17 cells. As notedabove, inhibition or reduction of IL-17 (and IL-22 or IL-21) leads tothe reduction of other proinflammatory molecules, thereby reducing orinhibitng inflammation.

Inhibiting Th1 and Th17 Responses

The disclosed C1ORF32 polypeptide selected from the group consisting ofSEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusionprotein or a conjugate thereof can be used to inhibit both the Th1 andTh17 pathways simultaneously. Using one anti-inflammatory agent toinhibit two separate pathways provides more robust inhibition orreduction of the immune response.

Promoting Th2 Responses and IL-10 Production.

Inflammation can also be treated by administering C1ORF32 polypeptideselected from the group consisting of SEQ ID NOs: 29, 30, 41-105, or afragment, variant, a homolog, a fusion protein or a conjugate thereofthereof to a subject in an amount effective to enhance Th2 responses,and the suppressive activity of IL-10 producing cells, and to enhancesuppressive or modulatory activity on the Th1 and/or Th17 pathways. Inthis embodiment the disclosed C1ORF32 polypeptide selected from thegroup consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant,a homolog, a fusion protein or a conjugate thereof cause an increasedsuppressive effect on IFN-gamma and/or IL-17 production. Anotherembodiment provides a method for treating inflammation by administeringan effective amount of C1ORF32 polypeptide selected from the groupconsisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, ahomolog, a fusion protein or a conjugate thereof to increase productionof IL-10 by Th2, Tregs or other immune cells.

Increased production of IL-10 results in the decreased production ofIL-17 by Th17 cells and deceased production of IFN-gamma by Th1 cells.In this embodiment, the C1ORF32 polypeptide selected from the groupconsisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, ahomolog, a fusion protein or a conjugate thereof can interact directlywith with immune cells to increase IL-10 production.

Still another embodiment provides a method for treating inflammation byadministering an effective amount of C1ORF32 polypeptide selected fromthe group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,variant, a homolog, a fusion protein or a conjugate thereof to inhibitor interfere with the Th1 pathway and Th17 pathway, and to enhance thesuppressive effect on the Th1 and/or Th17 pathways by Th2 cells.

The C1ORF32 polypeptide selected from the group consisting of SEQ IDNOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusion proteinor a conjugate thereof can also be administered to a subject in anamount effective to increase Th2 cell populations or numbers.

IL-10 production can be increased relative to a control by contactingTh2 cells, Tregs or other immune cells with an effective amount ofC1ORF32 polypeptide selected from the group consisting of SEQ ID NOs:29, 30, 41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof having C1ORF32 activity. The increase can occur invitro or in vivo.

Inflammatory Disease to be Treated

Representative inflammatory or autoimmune diseases and disorders thatmay be treated using C1ORF32 polypeptide selected from the groupconsisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, ahomolog, a fusion protein or a conjugate thereof include, but are notlimited to multiple sclerosis, rheumatoid arthritis, type I diabetes,psoriasis, systemic lupus erythematosus, inflammatory bowel disease,uveitis, and Sjogren's syndrome.

C1ORF32 polypeptide selected from the group consisting of SEQ ID NOs:29, 30, 41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof acts at multiple points in the inflammatory pathwaymaster regulator to control the expression and/or activity of effectorycytokines such as IFN-gamma and TNF-alpha. Therefore, the C1ORF32compositions described herein are particularly useful for treatingpatients that do not respond to TNF-alpha blockers such as Enbrel,Remicade, Cimzia and Humira, or where TNF-alpha blockers are not safe oreffective. In addition, because of its activity as a master regulator inthe inflammatory pathway, the C1ORF32 compositions disclosed areparticularly useful for treating chronic and persistent inflammation.

In a further embodiment, the C1ORF32 compositions described herein areused to treat relapsing and/or remitting multiple sclerosis.

Inhibition of Epitope Spreading

Epitope spreading refers to the ability of B and T cell immune responseto diversify both at the level of specificity, from a single determinantto many sites on an auto antigen, and at the level of V gene usage(Monneaux, F. et al., Arthritis &amp; Rheumatism, 46(6): 1430-1438(2002). Epitope spreading is not restricted to systemic autoimmunedisease. It has been described in T cell dependent organ specificdiseases such as Diabetes mellitus type 1 and multiple sclerosis inhumans, and EAE induced experimental animals with a variety of myelinproteins.

Epitope spreading involves the acquired recognition of new epitopes inthe same self molecule as well as epitopes residing in proteins that areassociated in the same macromolecular complex. Epitope spreading can beassessed by measuring delayed-type hypersensitivity (DTH) responses,methods of which are known in the art.

One embodiment provides a method for inhibiting or reducing epitopespreading in a subject by administering to the subject an effectiveamount of C1ORF32 polypeptide selected from the group consisting of SEQID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusionprotein or a conjugate thereof. In a further embodiment the C1ORF32polypeptide selected from the group consisting of SEQ ID NOs: 29, 30,41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof inhibits epitope spreading in individuals withmultiple sclerosis. Preferably, the C1ORF32 polypeptide selected fromthe group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,variant, a homolog, a fusion protein or a conjugate thereof inhibits orblocks multiple points of the inflammation pathway.

Yet another embodiment provides a method for inhibiting or reducingepitope spreading in subjects with multiple sclerosis by administeringto a subject an effective amount of C1ORF32 polypeptide selected fromthe group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,variant, a homolog, a fusion protein or a conjugate thereof to inhibitor reduce differentiation of, proliferation of, activity of, and/orcytokine production and/or secretion by Th1, Th17, Th22, and/or othercells that secrete, or cause other cells to secrete, inflammatorymolecules, including, but not limited to, IL-1beta, TNF-alpha, TGF-beta,IFN-gamma, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs. Anotherembodiment provides a method for treating multiple sclerosis byadministering to a subject an effective amount of C1ORF32 polypeptideselected from the group consisting of SEQ ID NOs: 29, 30, 41-105, or afragment, variant, a homolog, a fusion protein or a conjugate thereofthereof to interact with Tregs, enhance Treg activity, promote orenhances IL-10 secretion by Tregs, increase the number of Tregs,increase the suppressive capacity of Tregs, or combinations thereof.Another embodiment provides a method for treating multiple sclerosis byadministering to a subject an effective amount of C1ORF32 polypeptideselected from the group consisting of SEQ ID NOs: 29, 30, 41-105, or afragment, variant, a homolog, a fusion protein or a conjugate thereof tointeract with Th2 cells, enhance Th2 activity, promote or enhance IL-10secretion by Th2 cells, increase the number of Th2 cells, increase themodulatory capacity of Th2 cells, or combinations thereof.

Induction of Immune Tolerance

In one embodiment, the present invention provides a method for inducingor re-establishing immune tolerance in a subject by administering to thesubject an effective amount of C1ORF32 polypeptide selected from thegroup consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant,a homolog, a fusion protein or a conjugate thereof. In a furtherembodiment the C1ORF32 polypeptide selected from the group consisting ofSEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusionprotein or a conjugate thereof induces tolerance in individuals withimmune related diseases. In a specific embodiment the C1ORF32polypeptide, fragment or fusion protein thereof induces tolerance inindividuals with multiple sclerosis or any other immune related diseaseas described herein. Preferably, the C1ORF32 polypeptide selected fromthe group consisting of SEQ ID NOs: 29, 30, 41-105, or a fragment,variant, a homolog, a fusion protein or a conjugate thereof inhibits orblocks multiple points of the inflammation pathway. In another specificembodiment, the C1ORF32 polypeptide selected from the group consistingof SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, afusion protein or a conjugate thereof induces tolerance in individualswith rheumatoid arthritis. Another embodiment provides a method fortreating immune related diseases by administering to a subject aneffective amount of C1ORF32 polypeptide selected from the groupconsisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, ahomolog, a fusion protein or a conjugate thereof to induce immunetolerance by interacting with Tregs, enhancing Treg activity, increasingthe number of Tregs, increase the suppressive capacity of Tregs, orcombinations thereof. Another embodiment provides a method for treatingimmune related diseases by administering to a subject an effectiveamount of C1ORF32 polypeptide selected from the group consisting of SEQID NOs: 29, 30, 41-105, or a fragment, variant, a homolog, a fusionprotein or a conjugate thereof to promote or enhance IL-10 secretion byimmune cells.

Use of the Therapeutic Agents According to at Least Some Embodiments ofthe Invention for Adoptive Immunotherapy:

One of the cardinal features of some models of tolerance is that oncethe tolerance state has been established, it can be perpetuated to naiverecipients by the adoptive transfer of donor-specific regulatory cells.Such adoptive transfer studies have also addressed the capacity ofT-cell subpopulations and non-T cells to transfer tolerance. Suchtolerance can be induced by blocking costimulation or upon engagement ofa co-inhibitory B7 with its counter receptor. This approach, that havebeen successfully applied in animals and is evaluated in clinical trialsin humans, (Scalapino K J and Daikh D I. PLoS One. 2009; 4(6):e6031;Riley et al., Immunity. 2009; 30(5): 656-665) provides a promisingtreatment option for autoimmune disorders and transplantation.Therapeutic agents according to at least some embodiments of theinvention, are used for_for adoptive immunotherapy. Thus, in at leastsome embodiments, the invention provides methods for in vivo or ex vivotolerance induction, comprising administering effective amount of thetherapeutic agent according to at least some embodiments, to a patientor to leukocytes isolated from the patient, in order to inducedifferentiation of tolerogenic regulatory cells; followed by ex-vivoenrichment and expansion of said cells and reinfusion of the tolerogenicregulatory cells to said patient.

Alternatively, immune responses can be enhanced in a patient by removingimmune cells from the patient, contacting immune cells in vitro with anagent that inhibits C1ORF32 activity, and/or which inhibits theinteraction of C1ORF32 with its natural binding partners, andreintroducing the in vitro stimulated immune cells into the patient. Inanother embodiment, a method of modulating immune responses involvesisolating immune cells from a patient, transfecting them with a nucleicacid molecule encoding a form of C1ORF32, such that the cells expressall or a portion of the C1ORF32 polypeptide according to variousembodiments of the present invention on their surface, and reintroducingthe transfected cells into the patient. The transfected cells have thecapacity to modulate immune responses in the patient.

Pharmaceutical Compositions

The present invention, in some embodiments, features a pharmaceuticalcomposition comprising a therapeutically effective amount of atherapeutic agent according to the present invention. According to thepresent invention the therapeutic agent could be any one of solubleC1ORF32 polypeptide selected from the group consisting of SEQ ID NOs:29, 30, 41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof or a corresponding nucleic acid sequence encoding. Thepharmaceutical composition according to the present invention is furtherused for the treatment of immune related disorder and/or infection asdescribed herein. The therapeutic agents of the present invention can beprovided to the subject alone, or as part of a pharmaceuticalcomposition where they are mixed with a pharmaceutically acceptablecarrier.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., soluble C1ORF32polypeptide selected from the group consisting of SEQ ID NOs: 29, 30,41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof or a corresponding nucleic acid sequence encoding. Thepharmaceutical compounds according to at least some embodiments of thepresent invention may include one or more pharmaceutically acceptablesalts. A “pharmaceutically acceptable salt” refers to a salt thatretains the desired biological activity of the parent compound and doesnot impart any undesired toxicological effects (see e.g., Berge, S. M.,et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts includeacid addition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition according to at least some embodiments ofthe present invention also may include one or more pharmaceuticallyacceptable anti-oxidants. Examples of pharmaceutically acceptableantioxidants include: (1) water soluble antioxidants, such as ascorbicacid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,sodium sulfite and the like; (2) oil-soluble antioxidants, such asascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, andthe like; and (3) metal chelating agents, such as citric acid,ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,phosphoric acid, and the like. A pharmaceutical composition according toat least some embodiments of the present invention also may includeadditives such as detergents and solubilizing agents (e.g., TWEEN 20(polysorbate-20), TWEEN 80 (polysorbate-80)) and preservatives (e.g.,Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,mannitol). Examples of suitable aqueous and nonaqueous carriers that maybe employed in the pharmaceutical compositions according to at leastsome embodiments of the present invention include water, buffered salineof various buffer content (e.g., Tris-HCl, acetate, phosphate), pH andionic strength, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate.

Proper fluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsaccording to at least some embodiments of the present invention iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin. Sterile injectable solutionscan be prepared by incorporating the active compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about I percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms according to at least some embodiments of thepresent invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

A composition of the present invention can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for therapeutic agentsaccording to at least some embodiments of the present invention includeintravascular delivery (e.g. injection or infusion), intravenous,intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, oral,enteral, rectal, pulmonary (e.g. inhalation), nasal, topical (includingtransdermal, buccal and sublingual), intravesical, intravitreal,intraperitoneal, vaginal, brain delivery (e.g. intra-cerebroventricular,intra-cerebral, and convection enhanced diffusion), CNS delivery (e.g.intrathecal, perispinal, and intra-spinal) or parenteral (includingsubcutaneous, intramuscular, intraperitoneal, intravenous (IV) andintradermal), transdermal (either passively or using iontophoresis orelectroporation), transmucosal (e.g., sublingual administration, nasal,vaginal, rectal, or sublingual), administration or administration via animplant, or other parenteral routes of administration, for example byinjection or infusion, or other delivery routes and/or forms ofadministration known in the art. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion or using bioerodible inserts, and can be formulated in dosageforms appropriate for each route of administration. In a specificembodiment, a protein, a therapeutic agent or a pharmaceuticalcomposition according to at least some embodiments of the presentinvention can be administered intraperitoneally or intravenously.

Compositions of the present invention can be delivered to the lungswhile inhaling and traverse across the lung epithelial lining to theblood stream when delivered either as an aerosol or spray driedparticles having an aerodynamic diameter of less than about 5 microns. Awide range of mechanical devices designed for pulmonary delivery oftherapeutic products can be used, including but not limited tonebulizers, metered dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art. Some specific examples ofcommercially available devices are the Ultravent nebulizer (MallinckrodtInc., St. Louis, Mo.); the Acorn II nebulizer (Marquest MedicalProducts, Englewood, Colo.); the Ventolin metered dose inhaler (GlaxoInc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler(Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkind all haveinhalable insulin powder preparations approved or in clinical trialswhere the technology could be applied to the formulations describedherein.

In some in vivo approaches, the compositions disclosed herein areadministered to a subject in a therapeutically effective amount. As usedherein the term “effective amount” or “therapeutically effective amount”means a dosage sufficient to treat, inhibit, or alleviate one or moresymptoms of the disorder being treated or to otherwise provide a desiredpharmacologic and/or physiologic effect. The precise dosage will varyaccording to a variety of factors such as subject-dependent variables(e.g., age, immune system health, etc.), the disease, and the treatmentbeing effected. For the polypeptide compositions disclosed herein andnucleic acids encoding the same, as further studies are conducted,information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age, and generalhealth of the recipient, will be able to ascertain proper dosing. Theselected dosage depends upon the desired therapeutic effect, on theroute of administration, and on the duration of the treatment desired.For polypeptide compositions, generally dosage levels of 0.0001 to 100mg/kg of body weight daily are administered to mammals and more usually0.001 to 20 mg/kg. For example dosages can be 0.3 mg/kg body weight, 1mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kgbody weight or within the range of 1-10 mg/kg. An exemplary treatmentregime entails administration once per week, once every two weeks, onceevery three weeks, once every four weeks, once a month, once every 3months or once every three to 6 months. Generally, for intravenousinjection or infusion, dosage may be lower. Dosage regimens are adjustedto provide the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms according to at least some embodiments of thepresent invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

Optionally the polypeptide formulation may be administered in an amountbetween 0.0001 to 100 mg/kg weight of the patient/day, preferablybetween 0.001 to 20.0 mg/kg/day, according to any suitable timingregimen. A therapeutic composition according to at least someembodiments according to at least some embodiments of the presentinvention can be administered, for example, three times a day, twice aday, once a day, three times weekly, twice weekly or once weekly, onceevery two weeks or 3, 4, 5, 6, 7 or 8 weeks. Moreover, the compositioncan be administered over a short or long period of time (e.g., 1 week, 1month, 1 year, 5 years).

Alternatively, therapeutic agent can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of thetherapeutic agent in the patient. In general, the half-life for fusionproteins may vary widely. The dosage and frequency of administration canvary depending on whether the treatment is prophylactic or therapeutic.In prophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

A “therapeutically effective dosage” of C1ORF32 soluble proteinpolypeptide selected from the group consisting of SEQ ID NOs: 29, 30,41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof, preferably results in a decrease in severity ofdisease symptoms, an increase in frequency and duration of diseasesymptom-free periods, an increase in lifepan, disease remission, or aprevention or reduction of impairment or disability due to the diseaseaffliction.

One of ordinary skill in the art would be able to determine atherapeutically effective amount based on such factors as the subject'ssize, the severity of the subject's symptoms, and the particularcomposition or route of administration selected.

In certain embodiments, the polypeptide compositions are administeredlocally, for example by injection directly into a site to be treated.Typically, the injection causes an increased localized concentration ofthe polypeptide compositions which is greater than that which can beachieved by systemic administration. For example, in the case of aneurological disorder like Multiple Sclerosis, the protein may beadministered locally to a site near the CNS. In another example, as inthe case of an arthritic disorder like Rheumatoid Arthritis, the proteinmay be administered locally to the synovium in the affected joint. Thepolypeptide compositions can be combined with a matrix as describedabove to assist in creating a increased localized concentration of thepolypeptide compositions by reducing the passive diffusion of thepolypeptides out of the site to be treated.

Pharmaceutical compositions of the present invention may be administeredwith medical devices known in the art. For example, in an optionalembodiment, a pharmaceutical composition according to at least someembodiments of the present invention can be administered with a needleshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicaments through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in an optional embodiment, a therapeuticcomposition according to at least some embodiments of the presentinvention can be administered with a needles hypodermic injectiondevice, such as the devices disclosed in U.S. Pat. Nos. 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556.Examples of well-known implants and modules useful in the presentinvention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicaments through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

In certain embodiments, C1ORF32 polypeptide selected from the groupconsisting of SEQ ID NOs: 29, 30, 41-105, or a fragment, variant, ahomolog, a fusion protein or a conjugate thereof according to at leastsome embodiments of the present invention can be formulated to ensureproper distribution in vivo. For example, the blood-brain barrier (BBB)excludes many highly hydrophilic compounds. To ensure that thetherapeutic compounds according to at least some embodiments of thepresent invention cross the BBB (if desired), they can be formulated,for example, in liposomes. For methods of manufacturing liposomes, see,e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomesmay comprise one or more moieties which are selectively transported intospecific cells or organs, thus enhance targeted drug delivery (see,e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplarytargeting moieties include folate or biotin (see, e.g., U.S. Pat. No.5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem.Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995)FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother.39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. JPhysiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem.269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.

Formulations for Parenteral Administration

In a further embodiment, compositions disclosed herein, including thosecontaining peptides and polypeptides, are administered in an aqueoussolution, by parenteral injection. The formulation may also be in theform of a suspension or emulsion. In general, pharmaceuticalcompositions are provided including effective amounts of a peptide orpolypeptide, and optionally include pharmaceutically acceptablediluents, preservatives, solubilizers, emulsifiers, adjuvants and/orcarriers. Such compositions optionally include one or more for thefollowing: diluents, sterile water, buffered saline of various buffercontent (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; andadditives such as detergents and solubilizing agents (e.g., TWEEN 20(polysorbate-20), TWEEN 80 (polysorbate-80)), anti-oxidants (e.g., watersoluble antioxidants such as ascorbic acid, sodium metabisulfite,cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodiumsulfite; oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol; and metal chelating agents, such as citricacid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,phosphoric acid), and preservatives (e.g., Thimersol, benzyl alcohol)and bulking substances (e.g., lactose, mannitol). Examples ofnon-aqueous solvents or vehicles are ethanol, propylene glycol,polyethylene glycol, vegetable oils, such as olive oil and corn oil,gelatin, and injectable organic esters such as ethyl oleate. Theformulations may be freeze dried (lyophilized) or vacuum dried andredissolved/resuspended immediately before use. The formulation may besterilized by, for example, filtration through a bacteria retainingfilter, by incorporating sterilizing agents into the compositions, byirradiating the compositions, or by heating the compositions.

Formulations for Topical Administration

C1ORF32 polypeptide selected from the group consisting of SEQ ID NOs:29, 30, 41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof, nucleic acids, and vectors disclosed herein can beapplied topically. Topical administration does not work well for mostpeptide formulations, although it can be effective especially if appliedto the lungs, nasal, oral (sublingual, buccal), vaginal, or rectalmucosa.

Compositions can be delivered to the lungs while inhaling and traverseacross the lung epithelial lining to the blood stream when deliveredeither as an aerosol or spray dried particles having an aerodynamicdiameter of less than about 5 microns.

A wide range of mechanical devices designed for pulmonary delivery oftherapeutic products can be used, including but not limited tonebulizers, metered dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art. Some specific examples ofcommercially available devices are the Ultravent nebulizer (MallinckrodtInc., St. Louis, Mo.); the Acorn II nebulizer (Marquest MedicalProducts, Englewood, Colo.); the Ventolin metered dose inhaler (GlaxoInc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler(Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkind all haveinhalable insulin powder preparations approved or in clinical trialswhere the technology could be applied to the formulations describedherein.

Formulations for administration to the mucosa will typically be spraydried drug particles, which may be incorporated into a tablet, gel,capsule, suspension or emulsion. Standard pharmaceutical excipients areavailable from any formulator. Oral formulations may be in the form ofchewing gum, gel strips, tablets or lozenges.

Transdermal formulations may also be prepared. These will typically beointments, lotions, sprays, or patches, all of which can be preparedusing standard technology. Transdermal formulations will require theinclusion of penetration enhancers.

Controlled Delivery Polymeric Matrices

C1ORF32 polypeptide selected from the group consisting of SEQ ID NOs:29, 30, 41-105, or a fragment, variant, a homolog, a fusion protein or aconjugate thereof, nucleic acids, and vectors disclosed herein may alsobe administered in controlled release formulations. Controlled releasepolymeric devices can be made for long term release systemicallyfollowing implantation of a polymeric device (rod, cylinder, film, disk)or injection (microparticles). The matrix can be in the form ofmicroparticles such as microspheres, where peptides are dispersed withina solid polymeric matrix or microcapsules, where the core is of adifferent material than the polymeric shell, and the peptide isdispersed or suspended in the core, which may be liquid or solid innature. Unless specifically defined herein, microparticles,microspheres, and microcapsules are used interchangeably. Alternatively,the polymer may be cast as a thin slab or film, ranging from nanometersto four centimeters, a powder produced by grinding or other standardtechniques, or even a gel such as a hydrogel.

Either non-biodegradable or biodegradable matrices can be used fordelivery of polypeptides or nucleic acids encoding the polypeptides,although biodegradable matrices are preferred. These may be natural orsynthetic polymers, although synthetic polymers are preferred due to thebetter characterization of degradation and release profiles. The polymeris selected based on the period over which release is desired. In somecases linear release may be most useful, although in others a pulserelease or “bulk release” may provide more effective results. Thepolymer may be in the form of a hydrogel (typically in absorbing up toabout 90% by weight of water), and can optionally be crosslinked withmultivalent ions or polymers.

The matrices can be formed by solvent evaporation, spray drying, solventextraction and other methods known to those skilled in the art.Bioerodible microspheres can be prepared using any of the methodsdeveloped for making microspheres for drug delivery, for example, asdescribed by Mathiowitz and Langer, J. Controlled Release, 5:13-22(1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); andMathiowitz, et al., J. Appl Polymer ScL, 35:755-774 (1988). The devicescan be formulated for local release to treat the area of implantation orinjection —which will typically deliver a dosage that is much less thanthe dosage for treatment of an entire body—or systemic delivery. Thesecan be implanted or injected subcutaneously, into the muscle, fat, orswallowed.

Diagnostic Uses of C1ORF32

Soluble polypeptides according to at least some embodiments of thepresent invention may also be modified with a label capable of providinga detectable signal, either directly or indirectly, including, but notlimited to, radioisotopes and fluorescent compounds. Such labeledpolypeptides can be used for various uses, including but not limited to,prognosis, prediction, screening, early diagnosis, determination ofprogression, therapy selection and treatment monitoring of diseaseand/or an indicative condition, as detailed above.

According to at least some embodiments, the present invention provides amethod for imaging an organ or tissue, the method comprising: (a)administering to a subject in need of such imaging, a labeledpolypeptide; and (b) detecting the labeled polypeptide to determinewhere the labeled polypeptide is concentrated in the subject. When usedin imaging applications, the labeled polypeptides according to at leastsome embodiments of the present invention typically have an imagingagent covalently or noncovalently attached thereto. Suitable imagingagents include, but are not limited to, radionuclides, detectable tags,fluorophores, fluorescent proteins, enzymatic proteins, and the like.One of skill in the art will be familiar with other methods forattaching imaging agents to polypeptides. For example, the imaging agentcan be attached via site-specific conjugation, e.g., covalent attachmentof the imaging agent to a peptide linker such as a polyarginine moietyhaving five to seven arginines present at the carboxyl-terminus of andFc fusion molecule. The imaging agent can also be directly attached vianon-site specific conjugation, e.g., covalent attachment of the imagingagent to primary amine groups present in the polypeptide. One of skillin the art will appreciate that an imaging agent can also be bound to aprotein via noncovalent interactions (e.g., ionic bonds, hydrophobicinteractions, hydrogen bonds, Van der Waals forces, dipole-dipole bonds,etc.).

In certain instances, the polypeptide is radiolabeled with aradionuclide by directly attaching the radionuclide to the polypeptide.In certain other instances, the radionuclide is bound to a chelatingagent or chelating agent-linker attached to the polypeptide. Suitableradionuclides for direct conjugation include, without limitation, 18 F,124 I, 125 I, 131 I, and mixtures thereof. Suitable radionuclides foruse with a chelating agent include, without limitation, 47 Sc, 64 Cu, 67Cu, 89 Sr, 86 Y, 87 Y, 90 Y, 105 Rh, 111 Ag, 111 In, 117m Sn, 149 Pm,153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 211 At, 212 Bi, and mixturesthereof. Preferably, the radionuclide bound to a chelating agent is 64Cu, 90 Y, 111 In, or mixtures thereof. Suitable chelating agentsinclude, but are not limited to, DOTA, BAD, TETA, DTPA, EDTA, NTA, HDTA,their phosphonate analogs, and mixtures thereof. One of skill in the artwill be familiar with methods for attaching radionuclides, chelatingagents, and chelating agent-linkers to polypeptides of the presentinvention. In particular, attachment can be conveniently accomplishedusing, for example, commercially available bifunctional linking groups(generally heterobifunctional linking groups) that can be attached to afunctional group present in a non-interfering position on thepolypeptide and then further linked to a radionuclide, chelating agent,or chelating agent-linker.

Non-limiting examples of fluorophores or fluorescent dyes suitable foruse as imaging agents include Alexa Fluor® dyes (Invitrogen Corp.;Carlsbad, Calif.), fluorescein, fluorescein isothiocyanate (FITC),Oregon Green™; rhodamine, Texas red, tetrarhodamine isothiocynate(TRITC), CyDye™ fluors (e.g., Cγ2, Cγ3, Cγ5), and the like.

Examples of fluorescent proteins suitable for use as imaging agentsinclude, but are not limited to, green fluorescent protein, redfluorescent protein (e.g., DsRed), yellow fluorescent protein, cyanfluorescent protein, blue fluorescent protein, and variants thereof(see, e.g., U.S. Pat. Nos. 6,403,374, 6,800,733, and 7,157,566).Specific examples of GFP variants include, but are not limited to,enhanced GFP (EGFP), destabilized EGFP, the GFP variants described inDoan et al., Mol. Microbiol., 55:1767-1781 (2005), the GFP variantdescribed in Crameri et al., Nat. Biotechnol., 14:315-319 (1996), thecerulean fluorescent proteins described in Rizzo et al., Nat.Biotechnol, 22:445 (2004) and Tsien, Annu. Rev. Biochem., 67:509 (1998),and the yellow fluorescent protein described in Nagal et al., Nat.Biotechnol., 20:87-90 (2002). DsRed variants are described in, e.g.,Shaner et al., Nat. Biotechnol., 22:1567-1572 (2004), and includemStrawberry, mCherry, morange, mBanana, mHoneydew, and mTangerine.Additional DsRed variants are described in, e.g., Wang et al., Proc.Natl. Acad. Sci. U.S.A., 101:16745-16749 (2004) and include mRaspberryand mPlum. Further examples of DsRed variants include mRFPmars describedin Fischer et al., FEBS Lett., 577:227-232 (2004) and mRFPruby describedin Fischer et al., FEBS Lett., 580:2495-2502 (2006).

In other embodiments, the imaging agent that is bound to a polypeptideaccording to at least some embodiments of the present inventioncomprises a detectable tag such as, for example, biotin, avidin,streptavidin, or neutravidin. In further embodiments, the imaging agentcomprises an enzymatic protein including, but not limited to,luciferase, chloramphenicol acetyltransferase, β-galactosidase,β-glucuronidase, horseradish peroxidase, xylanase, alkaline phosphatase,and the like.

Any device or method known in the art for detecting the radioactiveemissions of radionuclides in a subject is suitable for use in thepresent invention. For example, methods such as Single Photon EmissionComputerized Tomography (SPECT), which detects the radiation from asingle photon gamma-emitting radionuclide using a rotating gamma camera,and radionuclide scintigraphy, which obtains an image or series ofsequential images of the distribution of a radionuclide in tissues,organs, or body systems using a scintillation gamma camera, may be usedfor detecting the radiation emitted from a radiolabeled polypeptide ofthe present invention. Positron emission tomography (PET) is anothersuitable technique for detecting radiation in a subject. Miniature andflexible radiation detectors intended for medical use are produced byIntra-Medical LLC (Santa Monica, Calif.). Magnetic Resonance Imaging(MRI) or any other imaging technique known to one of skill in the art isalso suitable for detecting the radioactive emissions of radionuclides.Regardless of the method or device used, such detection is aimed atdetermining where the labeled polypeptide is concentrated in a subject,with such concentration being an indicator of disease activity.

Non-invasive fluorescence imaging of animals and humans can also providein vivo diagnostic information and be used in a wide variety of clinicalspecialties. For instance, techniques have been developed over the yearsfor simple ocular observations following UV excitation to sophisticatedspectroscopic imaging using advanced equipment (see, e.g.,Andersson-Engels et al., Phys. Med. Biol., 42:815-824 (1997)). Specificdevices or methods known in the art for the in vivo detection offluorescence, e.g., from fluorophores or fluorescent proteins, include,but are not limited to, in vivo near-infrared fluorescence (see, e.g.,Frangioni, Curr. Opin. Chem. Biol., 7:626-634 (2003)), the Maestro™ invivo fluorescence imaging system (Cambridge Research & Instrumentation,Inc.; Woburn, Mass.), in vivo fluorescence imaging using a flying-spotscanner (see, e.g., Ramanujam et al., IEEE Transactions on BiomedicalEngineering, 48:1034-1041 (2001), and the like.

Other methods or devices for detecting an optical response include,without limitation, visual inspection, CCD cameras, video cameras,photographic film, laser-scanning devices, fluorometers, photodiodes,quantum counters, epifluorescence microscopes, scanning microscopes,flow cytometers, fluorescence microplate readers, or signalamplification using photomultiplier tubes.

The present invention is further illustrated by the below examplesrelated to C1ORF32 antigen, its domains and expression data as well asprophetic examples describing the manufacture of fully human antibodiesthereto. This information and examples is illustrative and should not beconstrued as further limiting. The contents of all figures and allreferences, patents and published patent applications cited throughoutthis application are expressly incorporated herein by reference.

EXAMPLES Example 1 Design and Production of Mutated C1ORF32 ECDPolypeptides

This Example relates to production of ECD fragments.

Materials and Methods

C1ORF32-P8-ECD-mFC Production in CHO Cells:

To produce C1ORF32-P8-ECD-mFC in CHO cells, retrovector constructscontaining cDNA that code for the protein were transduced into CHOcells, and 3-4 rounds of transductions were carried out. Cell pool ofthe last transduction was grown in shake flasks by fed-batch production.Medium was harvested at 12-14 days when cell viability dropped to −50%.

The harvested medium was clarified by depth filtration followed by 0.2μm filter, and the clarified material was subjected to Protein Achromatography. The eluate, pH ˜3.7, was incubated with mixing for 60minutes at room temperature to facilitate viral inactivation,neutralized to pH ˜7.2 and filtered through a 0.2 μm filter. Finally,the solution was buffer exchanged to PBS pH 7.2, concentrated to thedesired protein concentration and filtered.

Results

SDS-PAGE analysis, under non-reducing conditions, ofC1ORF32-P8-V1-ECD-mFC produced in CHO cells, as described below,revealed three main bands: the expected MW of ˜100 kD, a band at MW of˜751(D, and a third one at MW of ˜55kD. SDS-PAGE analysis in reducedconditions revealed two main bands—one at the expected MW of ˜50kD andan additional of ˜301(D.

Western Blot analyses using anti-C1ORF32-P8-V1-ECD and anti-mFcdemonstrated the presence of mainly three protein species: the intactfull-size dimer consisting of two chains each with an C1ORF32 ECD and aFc (homodimer); a one-arm cleaved dimer consisting of one chain with anC1ORF32 ECD and a Fc and second chain with mainly the Fc (heterodimer);and a two-armed cleaved dimer composed of two chains with mainly the Fc(fully cleaved dimer).

N-terminal sequencing revealed a major cleavage site towards theC-terminus of the C1ORF32 ECD between amino acids F and A at positions179 and 180 of H19011_1_P8_V1 or H19011_1_P8 (Seq ID NOs: 4 or 5). Toprevent this cleavage the amino acids F and A at positions 179 and 180of H19011_1_P8_V1 or H19011_1_P8 (SEQ ID NOs: 4 or 5) were mutated asfollows: FA->GA, FA->AA, and FA->GG. The resulted C1ORF32 ECD fragmentsare represented by, for example, SEQ ID NOs: 64, 96, and 45,respectively.

Example 2 Production of FC-Fused C1ORF32 Proteins

The Fc-fused C1ORF32 ECD proteins were produced as follows:

Fc-fused C1ORF32 ECD (SEQ ID NO:108), corresponds to C1ORF32 ECD withoutsignal peptide (SEQ ID NO: 64) fused to mouse mIgG2a Fc (SEQ ID NO: 31)via GS_TEV_linker_SG (SEQ ID NO:113).

Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110) corresponds toC1ORF32 ECD-Delta DLLPSFAVEIM fragment without signal peptide (SEQ IDNO:51) fused to mouse mIgG2a Fc (SEQ ID NO: 31) via GS_TEV_linker_SG(SEQ ID NO:113).

C1ORF32 ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) corresponds toC1ORF32 ECD-Delta RTGLLADLLPSFAVEIM fragment without signal peptide (SEQID. NO:29) fused to mouse mIgG2a Fc (SEQ ID NO: 31) via GS_TEV_linker_SG[(SEQ ID NO:113).

All the fused proteins were produced using two service providers;Catalent and ExcellGene, each using its own production protocol asdescribed below:

Production by Catalent (Middleton, Wis., USA): Codon-optimized cDNAsequences encoding C1ORF32 ECD (SEQ ID NO:108), Fc-fused C1ORF32ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), and C1ORF32 ECD-DeltaRTGLLADLLPSFAVEIM (SEQ ID NO:112) were produced at Catalent in CHOcells, in pIRESpuro3 vector.

The cDNA sequence of the insert was verified by Catalent and was used toconstruct GPEx® retrovectors, followed by four rounds of retrovectortransduction into Catalent's “in-house” CHO—S cell line. A pooledpopulation was produced and expanded and gene copy index was 2.7. Cellculture supernatants were analyzed by Catalent's Fc ELISA assay andrelative productivity of the 4× transduced pool was 28 μg/ml.

The protein was produced in 5 L wave bioreactor, and purified accordingto their in-house process. The level of purity was estimated at >95%, bySDS PAGE and Coomassie staining (data not shown). The concentration ofpurified protein was measured by absorbance at A280 nm, and estimated at1.60 mg/ml (using an absorbance coefficient of 1.28 mg/ml). Endotoxinlevels were tested, and estimated at 0.25-0.5EU/ml. A total of ˜400 mgwere obtained from ˜10 L of cell pool.

Production by ExcellGene (Valais, Switzerland): Codon-optimized cDNAsequences encoding C1ORF32 ECD (SEQ ID NO:108), Fc-fused C1ORF32ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), and C1ORF32 ECD-DeltaRTGLLADLLPSFAVEIM (SEQ ID NO:112) were synthesized and subcloned intoExcellGene's proprietary expression vector (pXLG6), designed for highyield transient gene expression vectors. Expression vectors were thentransiently-transfected into Exellgene's CHOExpress™ host cells, andcells were cultured for 12-14 days in TubeSpin50® bioreactors. Viabilitywas determined daily, and productivity was assessed at the end of theculture by Western Blot analysis. A second transient transfection wasthen performed, and the resultant cells were cultured in 1-2 L to obtainthe required amount of protein. Culture supernatants were clarified andpurified on Protein A columns. Purified proteins were analyzed by astandard SDS-PAGE gel under standard conditions, for which results areshown in FIG. 2, and concentration was determined by A280. Proteins weretested for bioburden and endotoxin. In FIG. 2, lane 12 relates to theprotein having the sequence of SEQ ID NO:110, while lane 13 relates tothe protein having the sequence of SEQ ID NO:112.

Example 3 The Effect of Fc-Fused C1ORF32 ECD (Seq Id No:108), Fc-FusedC1ORF32 ECD-Delta DLLPSFAVEIM (Seq Id No:110), and C1ORF32 ECD-DeltaRTGLLADLLPSFAVEIM (Seq Id No:112) on TCR-Mediated Activation of MouseCD4 T Cells.

The effect of Fc-fused C1ORF32 ECD (SEQ ID NO:108), Fc-fused C1ORF32ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), and C1ORF32 ECD-DeltaRTGLLADLLPSFAVEIM (SEQ ID NO:112)on TCR-mediated activation of mouse Tcells, was evaluated by testing their effect on IFNγ secretion and onthe expression of the activation marker CD69 in CD4+CD25-purified Tcells.

Methods Mouse CD4 T Cells Isolation.

CD4+CD25− T cells were isolated from pools of spleens and lymph nodes ofBALB/C mice by one step negative selection using T cell isolation Kit(Miltenyi Cat#130-093-227) according to the manufacturer's instructions.The purity obtained was >95%. The cells used in the functional assays,CD4+ T cells, were untouched CD4+CD25− cells, which includeCD4+CD62L^(high) naive T cells (85-90% of bulk CD4 population) andCD4+CD62L^(low) w memory cells.

Activation of Mouse CD4 T Cells

Anti-CD3 mAb (clone 145-2C11) alone or together with Fc-fused C1ORF32ECD (SEQ ID NO:108), Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ IDNO:110), and C1ORF32 ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) or withcontrol mIgG2a (BioXCell Cat. #BE0085) were co-immobilized at the statedconcentrations on 96-well flat bottom tissue culture plates (Sigma, Cat.#92096), overnight at 4° C. Wells were washed 3 times with PBS andplated with 1×10⁵ CD4+ T cells per well at 37° C. Culture supernatantswere collected 48 h post stimulation and analyzed for IFNγ secretionusing mouse IFNγ ELISA kit (R&D Systems). The effect of immobilizedFc-fused C1ORF32 ECD (SEQ ID NO:108), Fc-fused C1ORF32 ECD-DeltaDLLPSFAVEIM (SEQ ID NO:110), and C1ORF32 ECD-Delta RTGLLADLLPSFAVEIM(SEQ ID NO:112) (10 μg/ml) on the activation marker CD69 was analyzed byFACS, 48 h post simulations with plate bound anti-CD3.

Results Effect of Fc-fused C1ORF32 ECD (SEQ ID NO:108), Fc-fused C1ORF32ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), and C1ORF32 ECD-DeltaRTGLLADLLPSFAVEIM (SEQ ID NO:112) on Mouse CD4 T Cells IFNγ Secretion

In order to evaluate the effect of Fc-fused C1ORF32 ECD (SEQ ID NO:108),Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), and C1ORF32ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) on CD4 T cell response,titrated amounts of the respective Fc-fusion C1ORF32 ECD protein orcontrol mIgG2a were immobilized on 96-well plates together with anti-CD3mAb, and IFNγ secretion from CD4+ T cells was analyzed. Cells wereactivated for 48 hrs using full RPMI containing 10% FBS, 1 mM sodiumpyruvate, 100 IU/ml Pen-Strep (without 2-ME). The results presented inFIG. 3 indicate potent inhibitory effect Fc-fused C1ORF32 ECD (SEQ IDNO:108), Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), andC1ORF32 ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) on CD4 T cells IFN-γsecretion.

Effect of Fc-Fused C1ORF32 ECD (SEQ ID NO:108), Fc-Fused C1ORF32ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), and C1ORF32 ECD-DeltaRTGLLADLLPSFAVEIM (SEQ ID NO:112) on the Early Activation Marker CD69

In order to evaluate the effect of Fc-fused C1ORF32 ECD (SEQ ID NO:108),Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), and C1ORF32ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) on the early activationmarker CD69, an experiment similar to that described in FIG. 3 hereinwas carried out, using 10 ug/ml of Fc-fused C1ORF32 ECD (SEQ ID NO:108),Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), or C1ORF32ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112), respectively. All Fc-fusedC1ORF32 ECD proteins tested had a suppressive effect on early activationmarkers CD69 at 48 h post stimulation as manifested in a reduction ofCD69 upregulation upon stimulation, as shown in FIG. 4.

Conclusion

Immobilized Fc-fused C1ORF32 ECD (SEQ ID NO:108), Fc-fused C1ORF32ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), or C1ORF32 ECD-DeltaRTGLLADLLPSFAVEIM (SEQ ID NO:112) inhibit CD4 T cell activation to asimilar extent as manifested by reduced IFNγ secretion and CD69expression compare to control Ig upon TCR stimulation.

Example 4 The Effect of Fc-Fused C1ORF32 ECD (Seq Id No:108), Fc-FusedC1ORF32 ECD-DELTA DLLPSFAVEIM (Seq Id No:110), or C1ORF32 ECD-DeltaRTGLLADLLPSFAVEIM (Seq Id No:112) on Naive CD4+ T Cell Proliferation.Procedure:

Naive CD4⁺ T cells were isolated from spleens of D011.10 mice (Jackson)via automax sort: CD4-negative sort (Miltenyi Cat#130-095-248),including anti-CD25 (Miltenyi Cat#130-091-072) in the negative sortcocktail, followed by CD62L-positive sort (Miltenyi Cat #130-049-701).Balb/c total splenocytes were also collected from one mouse, andirradiated with 3000 rads to serve as antigen presenting cells (APCs)for the D011.10 CD4⁺ T cells. Naive CD4⁺ T cells were cultured at2.5×10⁵ cells per well in flat-bottom 96-well plates with irradiatedAPCs at a ratio of 1:1 (APCs to T cells) in 200 ul of HL-1 medium, andactivated with 2 ug/ml OVA323-339 in the presence of one of Fc-fusedC1ORF32 ECD (SEQ ID NO:108), (produced in CHO—S and in CHO-DG44 asdetailed in Example 2) Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ IDNO:110), (produced in CHO-DG44 as detailed in Example 2) and or C1ORF32ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) (produced in CHO—S and inCHO-DG44 as detailed in Example 3) or Isotype control Ig (mIgG2a,BioXCell Cat. # BE0085) at the indicated concentrations (0.1-10 ug/ml).The cells were pulsed with luCi of tritiated-thymidine at 24 hours, andharvested at 72 hours.

Results and Conclusions

As demonstrated in FIG. 5, the Fc-fused C1ORF32 ECD (SEQ ID NO:108), aswell as Fc-fused C1ORF32 ECD-Delta DLLPSFAVEIM (SEQ ID NO:110), orC1ORF32 ECD-Delta RTGLLADLLPSFAVEIM (SEQ ID NO:112) inhibit T cellproliferation in response to OVA323-339 activation in a dose dependentmanner A slightly reduced activity (approximately 3 fold) was achievedby the proteins produced in CHO-DG44 versus those produced in CHO—S.These differences reflect batch to batch variation of these proteins.

Example 5 The Effect of Fc-Fused C1ORF32 ECD (Seq Id No:108), Fc-FusedC1ORF32 ECD-Delta DLLPSFAVEIM (Seq Id No:110), or C1ORF32 ECD-DeltaRTGLLADLLPSFAVEIM (Seq Id No:112) on Autoimmune Disease or InfectiousDisease in a Subject

A plurality of animals, having a disease model appropriate for testingof the autoimmune disease, receives each of the above described Fc-fusedECD proteins in a plurality of doses. A dose dependent response is seen.

Similarly, a plurality of animals infected with an infectious disease inwhich T cell exhaustion is exhibited (for example through chronic viralinfection) and receives each of the above Fc-fused ECD proteins in aplurality of doses, in combination with an pharmaceutical agent in apharmaceutically effective amount against the infectious agent. A dosedependent response is seen.

Example 6

Effect of C1ORF32 Protein Fragments and/or Fusion Proteins on Activationof Naive CD4+ T Cells with Anti-CD3/Anti-CD28 Coated Beads

Naive CD4+ T cells are isolated from 5 SJL (Harlan, Israel) mice viaautomax sort. Beads are coated with anti-CD3 (0.5 ug/ml; clone 2C11) andanti-CD28 (2 ug/ml; clone 37.51 eBioscience) following manufacturer'sprotocol (Dynabeads M-450 Epoxy Cat. 140.11, Invitrogen), and withincreasing concentrations of any one of the C1ORF32 protein fragmentsand/or fusion proteins thereof (0.1-10 ug/ml). The total amount ofprotein used for beads coating with any one of the C1ORF32 proteinfragments and/or fusion proteins thereof is completed to 10 ug/ml withControl Ig. Naive CD4+ T cells (0.5×10⁶/well) are activated with thecoated beads at a ratio of 1:2 (beads to T cells). The cells are pulsedwith luCi of tritiated-thymidine after 24 hours, and harvested after 72.

The ability of any one of the C1ORF32 protein fragments and/or fusionproteins thereof to inhibite T cell proliferation and elicit its effectin a dose dependent manner is checked. An appropriate response is seen.

Example 7

Dose Response Effect of C1ORF32 Protein Fragments and/or Fusion ProteinsThereof on Mouse CD4+ T Cell Activation with Plate Bound Anti-CD3, asManifested in Cytokine Production and Expression of the ActivationMarker CD69.

Untouched CD4+CD25− T cells are isolated from pools of spleen and lymphnode cells of BALB/C mice by negative selection using CD4+CD62L+ T cellisolation Kit (Miltenyi Cat#130-093-227) according to the manufacturer'sinstructions. The desired purity obtained is >95%.

Tissue culture 96-well plates are coated overnight at 4° C. with 2 ug/mlanti-CD3 mAb (clone 145-2C11) in the presence of any one of the C1ORF32protein fragments and/or fusion proteins thereof at 1, 5 and 10 μg/ml.Control mIgG2a (Clone C1.18.4 from BioXCell; Cat#BE0085) is added toeach well in order to complete a total protein concentration of 12 μg/mlper well. Wells are plated with 1×10⁵ CD4+CD25− T cells per well. At 48hrs post stimulation, culture supernatants are collected and analyzedusing mouse IFNγ ELISA kit, and cells are analyzed for expression of theactivation marker CD69 by flow cytometry.

The inhibitory effects of any one of the C1ORF32 protein fragmentsand/or fusion proteins thereof on CD4 T cell activation is demonstratedby reduced IFNγ secretion and reduced expression of CD69 upon TCRstimulation, compared to control mIgG2a and CTLA4-Ig.

Example 8

The Effect of C1ORF32 Protein Fragments and/or Fusion Proteins Thereofon CD4+ T Cell Differentiation In Vitro.

To test the ability of any one of the C1ORF32 protein fragments and/orfusion proteins thereof to inhibit CD4+ T cell differentiation, naïveCD4+ T cells are isolated from D011.10 mice, which are transgenic for aT cell receptor (TCR) that is specific for OVA323-339 peptide. UsingD011.10 T cells enables studying both polyclonal (anti-CD3/anti-CD28mAbs) and peptide-specific responses on the same population of CD4+ Tcells. Naïve CD4+ T cells are isolated from D011.10 mice and activatedin culture in the presence of anti-CD3/anti-CD28 coated beads orOVA323-339 peptide plus irradiated BALB/c splenocytes, in the presenceof any one of the C1ORF32 protein fragments and/or fusion proteinsthereof, control Ig, or B7-H4 Ig. The cells are activated in thepresence of Th driving conditions as follows: Th0 cell-(IL-2), Th1cell-(IL-2+IL-12), Th2 cell-(IL-2+IL-4), or Th17cell-(TGF-β+IL-6+IL-23+anti-IL-2). The effects on T cell differentiationand Th-specific responses are assessed by measuring cell proliferationand subtype specific cytokine production: IL-4, IL-5, IL-10, IL-17,IFN-γ. An appropriate response is seen.

Example 9

Assessment of the Effect of any One of the C1ORF32 Protein Fragmentsand/or Fusion Proteins Thereof on Human T Cells Activation.

The effect of any one of the C1ORF32 protein fragments and/or fusionproteins thereof on human T cell response is tested by two different invitro assays using purified human T cells. In the first assay, human Tcells are activated by anti-CD3 and anti-CD28 coated beads, and in theother assay, activation is carried out using anti-CD3 and anti-CD28antibodies in the presence of autologous, irradiated PBMCs. Theregulatory activity of any one of the C1ORF32 protein fragments and/orfusion proteins thereof on human T cell activation, is evaluated bymeasuring cell proliferation and cytokine release.

Study I— Activation of Human T Cells with Anti-CD3 and Anti-CD28-CoatedBeads is Inhibited by Fusion Proteins

Naïve CD4+ T cells are isolated from 4 healthy human donors andactivated with anti-CD3 mAb/anti-CD28 mAb coated beads in the presenceof control mIgG2a, or any one of the C1ORF32 protein fragments and/orfusion proteins thereof. Two side-by-side culture sets are set up; oneculture being pulsed at 24 hours with tritiated-thymidine and harvestedat 72 hours while the second plate is harvested at 96 hours for cytokineproduction via LiquiChip.

Study II—Activation of Human T Cells with Irradiated Autologous PBMCs isInhibited by Fusion Proteins

Total PBMCs are isolated from fresh blood of healthy human donors usingficoll gradient. 10×10⁶ total PBMCs are resuspended in Ex-Vivo 20medium, and irradiated at 3000 rad. These cells are used to activate theisolated T cells in vitro, by presenting the anti-CD3, anti-CD28 andeither of the test proteins. The rest of PBMCs are used for isolation ofT cells using CD4+ T cell Isolation Kit II from Miltenyi.

For activation, 5×10⁵ isolated T cells are cultured in the presence of5×10⁵ autologous irradiate PBMCs. Anti-CD3 (0.5 μg/ml), anti-CD28 (2μg/ml) and either of any one of the C1ORF32 protein fragments and/orfusion proteins thereof or control Ig (mIgG2a) are added in a solubleform. The cultures are pulsed with luCi of triated thymidine at 24 hrs,and proliferation is measured at 72 hours.

Example 10 The Effect of any One of the C1ORF32 Protein Fragments UponEctopic Expression in APC-Like Cells, on Human T Cell Responses

The effects of any one of the C1ORF32 protein fragments on human T cellresponses are evaluated following their ectopic expression in ‘T cellstimulator’ cells: a murine thymoma cell line, Bw5147, which areengineered to express membrane-bound anti-human CD3 antibody fragments,that can trigger the TCR-complex on human T cells, with or withoutco-expression of putative costimulatory or coinhibitory ligands.

Codon-optimized cDNAs encoding any one of the C1ORF32 protein fragmentsare gene-synthesized and directionally cloned into a retroviral vectorpCJK2 via Sfi-I sites. Monocistronic expression constructs aregenerated. The constructs are validated by agarose gel electrophoresisand were expressed in Bw5147 cells displaying high levels of membranebound anti-CD3 antibody (Bw-3/2) (Leitner et al., 2010). As negativecontrol Bw5147 cells transduced with “empty” vector (pCJK2) are used. Inaddition, Bw-3/2 cells expressing costimulatory molecules (ICOSL andCD70) and Bw-3/2-cells expressing coinhibitory molecules (B7-H3 andB7-H1/PD-L1) are also used as controls. Homogenously high expression ofthe stimulating membrane-bound anti-CD3 antibody is confirmed by FACSusing a DyLight-649 anti-mouse IgG (H+L) antibody that reacts with themurine single chain antibody expressed on the stimulator cells. Presenceand high level transcription of expression monocistronic constructs inthe respective stimulator cells is confirmed by qPCR.

T cells are purified from buffy coats or heparinised blood derived fromhealthy volunteer donors and the mononuclear fraction is obtained bystandard density centrifugation using Ficoll-Paque (GE-Healthcare).Untouched bulk human T cells are obtained through MACS-depletion ofCD11b, CD14, CD16, CD19, CD33 and MHC-class II-bearing cells with therespective biotinylated mAb in conjunction with paramagneticstreptavidin beads (Leitner et al., 2009). Purified CD8 T cells and CD4T cells are obtained by adding biotinylated CD4 and CD8 mAb to thepools. Naïve CD4 T cells are isolated using the Naïve CD4+ T cellIsolation Kit II (Miltenyi Biotec). Following isolation, cells areanalyzed for purity by FACS, and samples with sufficient purity (>90%)were used for the experiments.

The stimulator cells are harvested, counted, irradiated (2×3000 rad) andseeded in flat-bottom 96-well plates (20000 cells/well). Liquid nitrogenstored MACS-purified T cells are thawed, counted and added to the wellsat 100.000 cells per well; total volume was 200 μl/well. Triplicatewells are set up for each condition. Following 48 hours of co-culture,³H-thymidine (final concentration of 0.025 mCi; PerkinElmer/NewEnglandNuclear Corporation, Wellesley, Mass.) are added to the wells. Followingfurther culturing for 18 hours, the plates were harvested onfilter-plates and incorporation of ³H-Thymidine was determined asdescribed in Pfistershammer et al., 2004. In addition, a series ofexperiments with MACS-purified T cell subsets (CD8 T cells, CD4 T cells,and naïve CD45RA-positive CD4 T cells) are performed. Additionalcontrols in all experiments include wells with stimulator cells alone toassess the cells microscopically and also to determine basal³H-Thymidine incorporation of the stimulator cell w/o T cells. Resultswith stimulator cells that quickly disintegrated following irradiationare excluded from the analysis.

Results show the effect of stimulator cells expressing any one of theC1ORF32 protein fragments on the proliferation of human bulk T cells,CD4+ T cells, CD8+ T cells, or naïve CD4 CD45RA+ T cells, compared tocells expressing control costimulatory molecules (ICOSL and CD70), whichresults in a consistent and pronounced stimulation of proliferation ofall cell subtypes, and compared to cells expressing control coinhibitorymolecules (B7-H3 and B7-H1/PD-L1), which results in a mild inhibition ofproliferation of different T cell subtypes.

Example 11

Characterizing the Target Cells for any One of the C1ORF32 ProteinFragments Proteins by Determining their Binding Profile to Immune Cells

Splenocytes from D011.10 mice (transgenic mice in which all of the CD4+T cells express a T cell receptor that is specific for OVA323-339peptide) are activated in the presence of OVA323-339 peptide, and cellsare collected at t=0, 6, 12, 24, and 48 hours following initialactivation to determine which cell type is expressing a receptor for anyone of the C1ORF32 protein fragments over time. Cells are thenco-stained for CD3, CD4, CD8, B220, CD19, CD11b, and CD11c.

Example 12

Assesment of the Effect of any One of the C1ORF32 Protein Fragmentsand/or Fusion Proteins Thereof on the Ability of B Cells to Class-Switchand Secrete Antibody

Resting B cells are isolated from unprimed C57BL/6 mice and activated invitro in the presence of anti-CD40 plus (i) no exogenous cytokine, (ii)IL-4, or (iii) IFN-γ. The cell cultures receive control Ig (mIgG2a),anti-CD86 mAb (as a positive control for increased Ig production), orany one of any one of the C1ORF32 protein fragments and/or fusionproteins thereof, at the time of culture set up, and are cultured for 5days. Any one of the C1ORF32 protein fragments and/or fusion proteinsthereof are tested at three concentrations each. At the end of culture,supernatants are tested for the presence of IgM, IgG1, and IgG2a viaELISA. If there appears to be an alteration in the ability of the Bcells to class-switch to one isotype of antibody versus another, thenthe number of B cells that have class switched is determined viaELISPOT. If there is an alteration in the number of antibody producingcells, then it is determined if there is an alteration in the level ofγ1- and γ2a-sterile transcripts versus the mature transcripts for IgG1and IgG2a. An appropriate response is seen.

Assesment of the Therapeutic Effect of any One of the C1ORF32 ProteinFragments and/or Fusion Proteins Thereof for Treatment of AutoimmuneDiseases

Example 13

Efficacy of any One of the C1ORF32 Protein Fragments and/or FusionProteins Thereof in Mouse R-EAE Model of Multiple Sclerosis

The therapeutic effect of any one of the C1ORF32 protein fragmentsand/or fusion proteins thereof for treatment of autoimmune diseases istested in a mouse model of Multiple Sclerosis; Relapsing RemittingExperimental Autoimmune Encephalomyelitis (R-EAE):

Female SJL mice 6 weeks old are purchased from Harlan and maintained inthe CCM facility for 1 week prior to beginning the experiment. Mice arerandomly assigned into groups of 10 animals and primed with 50 μgPLP139-151/CFA on day 0. Mice receive 6 i.p. injections of 100 ug/doseof any one of the C1ORF32 protein fragments and/or fusion proteinsthereof, mIgG2a isotype control, or CTLA4-Ig (mouse ECD fused to mouseIgG2a Fc) as positive control. Treatments begin at the time of onset ofdisease remission and are given 3 times per week for 2 weeks. Mice arefollowed for disease symptoms. On day 35, (during the peak of thedisease relapse) 5 mice of each group are assayed for DTH (delayed typehypersensitivity) response to disease inducing epitope (PLP139-151) andto relapse-associated myelin epitope (PLP178-191) via injection of 10 μgof PLP139-151 in one ear and PLP178-191 into the opposite ear. The levelof ear swelling is assayed at 24 hours post challenge.

The decrease in disease severity of R-EAE-induced mice upon treatmentwith any one of the C1ORF32 protein fragments and/or fusion proteinsthereof, in a therapeutic mode is tested and compared to the level ofinhibition of CTLA4-Ig.

In addition, inhibition of DTH responses to the disease inducing epitope(PLP139-151) and to relapse-associated epitope (PLP178-191). in R0EAEmice treated with any one of the C1ORF32 protein fragments and/or fusionproteins thereof is tested.

To test the dose dependency of the efficacy of any one of the C1ORF32protein fragments and/or fusion proteins thereof as well as its mode ofaction in the PLP-induced R-EAE model, disease is induced as describedabove and mice are treated from onset of disease remission with 100, 30or 10 ug/dose of any one of the C1ORF32 protein fragments and/or fusionproteins thereof, 3 times per week over two weeks. The ability ofC1ORF32 protein fragments and/or fusion proteins thereof to decrease thelevel of disease severity in a dose dependent manner, as well as theability to inhibit DTH responses to spread epitopes PLP178-191 andMBP84-104, to inhibit proliferation as well as reduction in IFNγ, IL-17,IL-4 and IL-10 release is tested.

The beneficial effect of any one of the C1ORF32 protein fragments and/orfusion proteins thereof in the R-EAE model on reduction in theinfiltration of immune cells to the CNS is also tested.

Example 14

Efficacy of any of the C1ORF32 Protein Fragments and/or Fusion ProteinsThereof in Mouse CIA Models of Rheumatoid Arthritis

Any one of the C1ORF32 protein fragments and/or fusion proteins thereofis tested in mouse model of collagen-induced arthritis (CIA) which is amodel of rheumatoid arthritis. Male DBA/1 mice are housed in groups of8-10, and maintained at 21° C.±2° C. on a 12 h light/dark cycle withfood and water ad libitum. Arthritis is induced by immunisation withtype II collagen emulsified in complete Freund's adjuvant. Mice aremonitored on a daily basis for signs of arthritis. On the appearance ofarthritis (day 1) treatment with any one of the C1ORF32 proteinfragments and/or fusion proteins thereof, mIgG2a isotype control orCTLA4-Ig (mouse ECD fused to mouse IgG2a Fc) as positive control (100ug/dose, each) is initiated and given 3 times per week for 10 days. Hindfootpad swelling is measured (using microcalipers), as well as thenumber and degree of joint involvement in all four limbs. This yieldstwo measurements, clinical score and footpad thickness that can be usedfor statistical assessment.

At the end of the treatment period mice are bled and sacrificed. Forhistological analysis, paws are removed at post mortem, fixed inbuffered formalin (10% v/v), then decalcified in EDTA in bufferedformalin (5.5% w/v). The tissues are then embedded in paraffin,sectioned and stained with haematoxylin and eosin. The scoring system isas follows: 0=normal; 1=synovitis but cartilage loss and bone erosionsabsent or limited to discrete foci; 2=synovitis and significant erosionspresent but normal joint architecture intact; 3=synovitis, extensiveerosions, joint architecture disrupted.

The ability of the treatment of mice with established CIA with any oneof the C1ORF32 protein fragments and/or fusion proteins thereof toresult in potent reduction of clinical score, paw swelling andhistological damage is tested and compared to the efficacy obtained withCTLA4-Ig.

Effect of any of the C1ORF32 Protein Fragments and/or Fusion ProteinsThereof on Tolerance Induction in Transfer Model of CIA

To further understand the effect of any one of the C1ORF32 proteinfragments and/or fusion proteins thereof on immune regulation, theability of these proteins to induce tolerance in a transfer model ofarthritis is analyzed.

In brief, spleen and LN cells from arthritic DBA/1 mice treated for 10days with any one of the C1ORF32 protein fragments and/or fusionproteins thereof or control Ig2a are removed and injected i.p intoT-cell deficient C.B-17 SCID recipients. The mice then receive aninjection of 100 μg type II collagen (without CFA), necessary forsuccessful transfer of arthritis. Arthritis is then monitored in theSCID mice. Histology is performed and anti-collagen antibody levels aremeasured to determine that the C1ORF32 protein fragments and/or fusionproteins thereof treatment confers long-term disease protection.

Example 15

Assessment of the Effect of any One of C1ORF32 Protein Fragments and/orFusion Proteins Thereof in a Viral Infection Model of TMEV

Theiler's murine encephalomyelitis virus (TMEV) is a natural endemicpathogen of mice that causes an induced demyelinating disease (TMEV-IDD)in susceptible strains of mice (SJL/J, H-2KS) that resembles the primaryprogressive form of MS (Munz et al., Nat Rev Immunol 2009; 9:246-58).TMEV infection results in a life-long persistent virus infection of theCNS leading to development of a chronic T cell-mediated autoimmunedemyelinating disease triggered via de novo activation of CD4 T cellresponses to endogenous myelin epitopes in the inflamed CNS (i.e.epitope spreading) (Miller et al., Nat Med 1997; 3:1133-6; Katz-Levy etal., J Clin Invest 1999; 104:599-610).

SJL mice clear the majority of the virus within 21 days post infection,however a latent viral infection is maintained and infect microglia,astrocytes, and neurons. Disease symptoms are manifested around day25-30 post infection.

The effect of treatment with any one of C1ORF32 protein fragments and/orfusion proteins thereof on acute and chronic phases of viral infectionis studied in the TMEV-IDD model by assessment of viral clearance anddisease severity.

Method:

Female SJL/J mice (5-6 weeks) are infected with TMEV by intracranialinoculation in the right cerebral hemisphere of 3×10⁷ plaque formingunits (PFU) of the BeAn strain 8386 of TMEV in 30 ul serum-free medium.From day 2 post infection mice are treated with Control Ig, or any oneof C1ORF32 protein fragments and/or fusion proteins thereof, at 100ug/dose each; 3 doses/week for 2 weeks.

Mice are followed for clinical scoring. On day 7 and day 14 postinfection (after 3 and 6 treatments respectively) brains and spinalcords are collected from 5 mice in each treatment group for plaqueassays. The tissues are weighted so that the ratio of PFU/mg of CNStissue could be calculated after the plaque assay is completed.

TMEV Plaque Assay:

Brains and spinal cords of mice treated with Control Ig (mouse IgG2a),or with any one of C1ORF32 protein fragments and/or fusion proteinsthereof are collected at days 7 and 14 post-infection from non-perfusedanesthetized mice. The Brains and spinal cords are weighed, andhomogenized. CNS homogenates are serially diluted in DMEM and added totissue culture-treated plates of confluent BHK-21 cells for 1 hincubation at room temperature, with periodic gentle rocking.

A media/agar solution is mixed 1:1 (volume:volume), added to cells andallowed to solidify at room temperature. The plates are then cultured at34 deg C. for 5 days. At the end of culture, 1 ml of formalin is addedand incubated at room temperature for 1 h to fix the BHK monolayer. Theformalin is poured off into a waste container, and the agar is removedfrom the plates. Plaques are visualized by staining with crystal violetfor 5 min, and plates are gently rinsed with diH2O. To determine PFU/mlhomogenate, the number of plaques on each plate is multiplied by thedilution factor of the homogenate and divided by the amount ofhomogenate added per plate. The PFU/ml is divided by the weight of thetissue to calculate PFU/mg tissue.

Example 16

Assessment of the Effect of any One of C1ORF32 Protein Fragments and/orFusion Proteins Thereof on Primary and Secondary Immune Response toViral Infection in a Mouse Model of Influenza

To test the effect of any one of C1ORF32 protein fragments and/or fusionproteins thereof on primary and secondary immune responses to viralinfection, BALB/c naïve mice (for primary immune responses) and‘HA-memory mice’, is used, as well as ‘polyclonal flu-memory mice’ (toassess secondary responses mediated by memory CD4 T cells), which aregenerated as detailed in Teijaro et al., J Immunol. 2009: 182;5430-5438, and described below.

To obtain ‘HA-memory mice’, first HA-specific memory CD4 T cells aregenerated, naive CD4 T cells are purified from spleens of HA-TCR miceWALB/c-HA mice which express transgenic T cell receptor (TCR) specificfor influenza hemagglutinin (HA) peptide (110-119)] and primed in vitroby culture with 5.0 microg/ml HA peptide and mitomycin C-treated,T-depleted BALB/c splenocytes as APCs for 3 days at 37° C. The resultantactivated HA-specific effector cells are transferred into congenicBALB/c (Thy1.1) hosts (5×10⁶ cells/mouse) to yield “HA-memory mice” witha stable population of HA-specific memory CD4 T cells.

To obtain ‘polyclonal-memory mice’, first polyclonal influenza-specificmemory CD4 T cells are generated, by infecting BALB/c mice intranasallywith a sublethal dose of PR8 influenza, CD4 T cells are isolated 2-4months postinfection, and the frequency of influenza-specific memory CD4T cells is determined by ELISPOT. CD4 T cells from previously primedmice are transferred into BALB/c hosts to generate “polyclonalflu-memory” mice with a full complement of endogenous T cells.

Primary and secondary responses to influenza virus are tested byinfecting naïve BALB/c mice or BALB/c-HA memory mice and BALB/c‘polyclonal flu-memory mice’ with sublethal or lethal doses of PR8influenza virus by intranasal administration.

Mice are treated with any one of C1ORF32 protein fragments and/or fusionproteins thereof or with mIgG2a control before and following influenzachallenge. Weight loss and mortality will be monitored daily. Six daysafter the challenge, viral content in the bronchoalveolar lavage (BAL)is analyzed by collecting lavage liquid and testing the supernatant forviral content by determining the tissue culture infectious dose 50%(TCID50) in MDCK cells. In addition, lung tissue histopathology isperformed.

To test the effect of any one of C1ORF32 protein fragments and/or fusionproteins thereof on T cell expansion BALB/c or BALB/c-HA memory mice orBALB/c ‘polyclonal flu-memory mice’ are infected as above andadministered with BrdU (1 mg/dose) on days 3, 4 and 5 post infection. Onday 6, spleen and lung are harvested and BrdU incorporation isestimated. Cytokine production by lung memory CD4 T cells duringinfluenza challenge is also studied in HA-specific memory CD4 T cellsstimulated in vitro with HA peptide in the presence of any one ofC1ORF32 protein fragments and/or fusion proteins thereof or IgG2a for 18hours.

Example 17

Assessment of the Effect of any One of C1ORF32 Protein Fragments and/orFusion Proteins Thereof on Primary and Secondary CD8 T Cell Response toViral Infection in a Mouse Model of Influenza

The effect of any one of C1ORF32 protein fragments and/or fusionproteins thereof on primary CD8 T cell responses to influenza virus isstudied according to methods as described in the literature (Hendriks etal., J Immunol 2005; 175; 1665-1676; Bertram et al., J Immunol. 2004;172:981-8) using C57BL/6 mice infected with influenza A HKx31 byintranasal or intraperitoneal administration. Any one of C1ORF32 proteinfragments and/or fusion proteins thereof or mIgG2a control isadministered during priming Animal weight loss and mortality ismonitored daily. To follow virus-specific CD8+ T cells, MHC H-2Dbtetramers loaded with the major CD8 T cell epitope, the NP₃₆₆₋₃₇₄peptide are used. Virus-specific H-2D^(b)/NP366-374+CD8+ T cells in thelung, draining lymph nodes, and spleen are expected to reach a peakaround day 8-10 post infection and decline thereafter to only 1.5%virus-specific CD8 T cells (Hendriks et al J Immunol 2005; 175;1665-1676; Bertram et al., J Immunol. 2002; 168:3777-85; Bertram et a.,J Immunol. 2004; 172:981-8). Thus, mice are sacrificed at days 8 and 21post infection, and virus-specific CD8 T cell numbers is evaluated inthe lung, draining lymph nodes and spleen. Viral clearance is assessed.CD8 T cell responses are evaluated in spleen cell suspensions, andinclude intracellular IFN-γ staining and CTL activity, as previouslydescribed (Bertram et a., J Immunol. 2004; 172:981-8) and detailedbelow.

Cells are surface-stained with FITC-conjugated anti-mouse CD62L,PE-conjugated anti-mouse CD8 to measure CD8+ activated T cells (oranti-mouse CD4 to follow CD4+ cells). In addition to these Abs,allophycocyanin-labeled tetramers consisting of murine class I MHCmolecule H-2D^(b), β₂-microglobulin, and influenza NP peptide, NP₃₆₆₋₃₇₄are used to measure influenza-specific CD8 T cells. For intracellularIFN-γ staining, cell suspensions are restimulated in culture medium for6 h at 37° C. with 1 μM NP366-374 peptide and GolgiStop (BD PharMingen,San Diego, Calif.). Cells are then harvested, resuspended in PBS/2%FCS/azide, and surface stained with PE-anti-CD8 and FITC-anti-CD62L asdescribed above. After surface staining, cells will be fixed inCytofix/Cytoperm solution (BD PharMingen) and then stained withallophycocyanin-conjugated antimouse IFN-γ diluted in 1× perm/washsolution (BD PharMingen). Samples are analyzed by Flow Cytometry.

For cytotoxicity assays (CTL responses) splenocytes frominfluenza-infected mice are incubated for 2 h at 37° C. to removeadherent cells. Serial 3-fold dilutions of effectors are assayed foranti-influenza NP₃₆₆₋₃₇₄-specific CTL activity against ⁵¹Cr-labeled EL4cells pulsed with 50 μM NP₃₆₆₋₃₇₄ peptide for 6 h as described byBertram et al 2002 and Bertram et al 2004.

At 3 weeks postinfection, some mice are rechallenged with theserologically distinct influenza A/PR8/34 (PR8), which shares the NPgene with influenza A HKx31, but differs in hemagglutinin andneuraminidase, so that neutralizing Abs do not limit the secondary CTLresponse. Mice are sacrificed at days 5 & 7 following virus rechallenge,and virus-specific CD8 T cell numbers is evaluated in the lung, draininglymph nodes and spleen as described by Hendriks et al and Bertram et al(Hendriks et al., J Immunol 2005; 175; 1665-1676; Bertram et al., JImmunol. 2004; 172:981-8) and detailed above. Secondary CD8 T cellresponses, including intracellular IFN-γ staining and CTL activity, areevaluated in spleen cell suspensions of mice at days 5 & 7 followingvirus rechallenge, as described above.

To determine the effect of any one of C1ORF32 protein fragments and/orfusion proteins thereof on expansion and accumulation of memory CD8+ Tcells during the secondary response, adoptive transfer experiments areperformed, according to methods previously described (Hendriks et al., JImmunol 2005; 175; 1665-1676; Bertram et al., J Immunol. 2004;172:981-8): mice are immunized with influenza influenza A HKx31.Twenty-one days later, T cells are purified from spleens on mouse T cellenrichment immunocolumns (Cedarlane Laboratories, Hornsby, Ontario,Canada) and labeled with CFSE (alternatively Thy1.1 congenic mice areused as recipients). Equal numbers of tetramer-positive T cells areinjected through the tail vein of recipient mice. Mice are rechallengedwith influenza virus as described above, and 7 days later splenocytesare evaluated for donor virus-specific CD8 T cells, as detailed above.

Example 18

Assessment of Protein Expression in Exhausted T Cells, and the Bindingand Effect of any One of C1ORF32 Protein Fragments and/or FusionProteins Thereof on Reversing Exhausted T Cell Phenotype

Memory CD8 T-cell differentiation proceeds along distinct pathways afteran acute versus a chronic viral infection (Klenerman and HillNat Immunol6, 873-879, 2005). Memory CD8 T cells generated after an acute viralinfection are highly functional and constitute an important component ofprotective immunity. In contrast, chronic infections are oftencharacterized by varying degrees of functional impairment ofvirus-specific T-cell responses, and this defect is a principal reasonfor the inability of the host to eliminate the persisting pathogen.Although functional effector T cells are initially generated during theearly stages of infection, they gradually lose function during thecourse of the chronic infection leading to exhausted phenotypecharacterized by impaired T cell functionality.

Study I. The Effect of any One of C1ORF32 Protein Fragments and/orFusion Proteins Thereof on Clearance of Viral Infection and on T CellFunctions During Acute and Chronic Viral Infection.

The effect of any one of C1ORF32 protein fragments and/or fusionproteins thereof on acute and chronic viral infection is evaluated in amouse model of infection with LCMV (lymphocytic chroriomeningitis virus)according to methodology described by Wherry et al J. Virol. 77:4911-4927, 2003 and Barber et al Nature, 2006, and detailed below.

Two LCMV strains that can cause either acute or chronic infections inadult mice are used; the Armstrong strain which is cleared within aweek, and the clone 13 strain which establishes a persistent infectionthat can last for months. As these two strains differ in only two aminoacids, preserving all known T cell epitopes, it is possible to track thesame CD8 T cell responses after an acute or chronic viral infection. Incontrast to the highly robust memory CD8 T cells generated after anacute Armstrong infection, LCMV-specific CD8 T cells become exhaustedduring a persistent clone 13 infection (Wherry et al J. Virol. 77:4911-4927, 2003; Barber et al., Nature. 2006; 439:682-7).

Mice are infected with 2×10⁵ PFU of Armstrong strain of LCMVintraperitoneally to initiate acute infection or 2×10⁶ PFU of C1-13intravenously to initiate chronic infection. Mice are treated i.p. withany one of C1ORF32 protein fragments and/or fusion proteins thereof orwith mIgG2a control, and with specific anti-C1ORF32 protein—antibody, oran isotype control.

The mice are monitored for numbers of virus specific CD8 T cells in thespleen, using virus-specific MHC tetramer epitopes, such asD^(b)NP₃₉₆₋₄₀₄ and D^(b)GP₃₃₋₄₁ which differ in acute or chronicinfections. CD8 T cell functional assays, such as intracellularcytokines levels and CTL activity, are carried out as described byWherry et al J. Virol. 77: 4911-4927, 2003, and similarly to thosedescribed in Example 40. Additional assays include production bysplenocytes after stimulation with virus specific epitopes; andassessment of viral titers in the serum and in the spleen, liver, lungand kidney (Wherry et al J. Virol. 77: 4911-4927, 2003; Barber et al.,Nature. 2006; 439:682-7).

Study II. Assessment of C1ORF32 Protein Expression on Exhausted T Cellsand Binding of any One of C1ORF32 Protein Fragments and/or FusionProteins Thereof to Exhausted T Cells in Order to Evaluate Regulation ofthese Proteins or their Counterpart Receptors During Exhaustion of TCells:

T cells are isolated from mice with chronic LCMV infection induced withC1-13 strain. The cells are co-stained with fluorescently labeledanti-PD-1 Ab as positive control (PD-1 is highly expressed by exhaustedT cells) and biotinylated C1ORF32 protein fragments and/or fusionproteins thereof or biotinylated anti-C1ORF32 protein antibodies, andrespective isotype control. Binding is detected by FACS analysis usingfluorescently labeled streptavidin.

Example 19 Assessment of any One of C1ORF32 Protein Fragments Expressionin Follicular Helper T (Tfh) Cells and the Binding of Ig Fusion Proteinsto TFH Cells

Follicular helper T (Tfh) cells are a subset of CD4+ T cells specializedin B cell help (reviewed by Crotty, Annu. Rev. Immunol. 29: 621-663,2011). Tfh cells migrate into B cell follicles within lymph nodes, andinteract with cognate B cells at the T cell-B cell border andsubsequently induce germinal center B cell differentiation and germinalcenter formation within the follicle (Reviewed by Crotty, Annu. Rev.Immunol. 29: 621-663, 2011). The requirement of Tfh cells for B cellhelp and T cell-dependent antibody responses, indicates that this celltype is of great importance for protective immunity against varioustypes of infectious agents, as well as for rational vaccine design.

Tfh cells are readily identifiable at the peak of the CD4+ T cellresponse to an acute lymphocytic choriomeningitis virus (LCMV) infectionas CXCR5^(hi)SLAM^(lo)BTLA^(hi)PD1^(hi)Bcl6⁺ virus-specific CD4+ T cells(Choi et al 2011, Immunity 34: 932-946). T cells are isolated from micewith acute LCMV infection induced with 2×10⁵ PFU of Armstrong strain ofLCMV administered intraperitoneally. The cells are co-stained withfluorescently labeled antibodies for markers of Tfh (CXCR5, PD1, BTLA,Bcl6) which are highly expressed by Tfh cells, and biotinylated C1ORF32protein fragments and/or fusion proteins thereof or biotinylatedantibodies specific for C1ORF32 proteins, and respective isotypecontrols. Binding of Fc fused protein or antibody is detected by FACSanalysis using fluorescently labeled streptavidin. An appropriateresponse is seen.

Example 20

Assessment of the Effect of any One of C1ORF32 Protein Fragments and/orFusion Proteins Thereof on Follicular Helper T (Tfh) Cells Generationand Activity

In order to investigate the effect of any one of C1ORF32 proteinfragments and/or fusion proteins thereof on Tfh differentiation anddevelopment of B cell immunity in vivo, C57BL/6 are treated with any oneof C1ORF32 protein fragments and/or fusion proteins thereof and anisotype control throughout the course of an acute viral infection withArmstrong strain of LCMV (lymphocytic choriomeningitis virus). Tfhdifferentiation and Bcl6 protein expression is assessed by FACS analysisas described by Eto et al 2011 (PLoS One 6: e17739). Splenocytes areanalyzed 8 days following LCMV infection, Tfhgeneration)(CD44^(hi)CXCR^(5hi)SLAM^(lo) and Bcl6 expression isevaluated by FACS analysis. In addition, the effect of any one ofC1ORF32 protein fragments and/or fusion proteins thereof onantigen-specific B cell responses is evaluated as described by Eto et al2011 (PLoS One 6: e17739), including titers of anti-LCMV IgG in theserum at 8 days following LCMV infection, and quantitation by FACSanalysis of plasma cell (CD138⁺IgD⁻) development at 8 dayspost-infection, gated on CD19+ splenocytes.

Example 21

The Effect of any One of C1ORF32 Protein Fragments and/or FusionProteins Thereof in Modulation of Type 1 Diabetes in Nod Mice, CD28-KONOD, and B7-2-KO NOD

The effect of any one of C1ORF32 protein fragments and/or fusionproteins thereof is studied in a widely used mouse model of type 1diabetes: nonobese diabetic (NOD) mice which develop spontaneous In NODmice, spontaneous insulitis, the hallmark pathologic lesion, evolvesthrough several characteristic stages that begin with peri-insulitis andend with with invading and destructive insulitis and overt diabetes.Peri-insulitis is first observed at 3-4 wk of age, invading insulitis at8-10 wk, and destructive insulitis appears just before the onset ofclinical diabetes, with the earliest cases at 10-12 wk. At 20 wk of age,70-80% of female NOD mice become diabetic (Ansari et al 2003 J. Exp.Med. 198: 63-69).

Two KO mice: CD-28-KO NOD mice and B7-1/B7-2 double KO NOD mice, —whichdevelop accelerated diabetes (Lenschow et al 1996 Immunity 5: 285-293;Salomon et al 2000 Immunity 12: 431-440), are also used.

Study I: NOD mice are treated with any one of C1ORF32 protein fragmentsand/or fusion proteins thereof early and late phases during theevolution of diabetes, before or after disease onset, to examine theeffects of these compounds on disease pathogenesis and to demonstratethat such treatment reduces disease onset and ameliorates pathogenesis.To study the effect on insulitis, blood glucose levels are measured 3times/week, for up to 25 weeks (Ansari et al 2003 J. Exp. Med. 198:63-69).

Mechanism of disease modification and mode of action is studied byexperimental evaluation of individual immune cell types: pancreas,pancreatic LNs and spleen will be harvested to obtain Tregs, Th subtypesand CD8 T cells, DCs and B cells. Effect on cytokines secretion fromcells isolated from pancreas, pancreatic LN and spleen is analysed,focused on IFNg, IL-17, IL-4, IL-10 and TGFb. Upon effect of the testedcompounds, the mechanism of disease modification is studied byexamination of individual immune cell types (including Tregs, Thsubtypes and CD8 T cells, DCs and B cells); cytokines (IFNg, IL-17,IL-4, IL-10 and TGFb) and histology. Histologycal analysis of thepancreas is carried out to compare the onset of insulitis, and thelymphocyte infiltration.

Study II—the Effect of any One of C1ORF32 Protein Fragments and/orFusion Proteins Thereof in Modulation of Type 1 Diabetes in AdoptiveTransfer Model

To further investigate the mode of action of the Ig fusion proteins, anadoptive transfer model of diabetes is used. T cells from diabetic orprediabetic NOD donors are transfered to NOD SCID recipient mice. Thesemice are monitored for development of diabetes. The urine glucose andblood glucose, and assess histology of the pancreas, and T cellresponses are monitored as described in the previous example.

Study III—Diabetes is also induced by the transfer of activatedCD4+CD62L+CD25-BDC2.5 T cells (transgenic for TCR recognizing isletspecific peptide 1040-p31 activated by incubation with 1040-p31) to NODrecipients. Mice are treated with any one of C1ORF32 protein fragmentsand/or fusion proteins thereof, control mIgG2a or positive control.Treatments begin 1 day following transfer. Mice are followed for glucoselevels 10-28 days post transfer (Bour-Jordan et al., J Clin Invest.2004; 114(7):979-87).

Seven days post treatment pancreas, spleen, pancreatic LN and peripherallymph node cells are extracted and examined for different immune cellpopulations. In addition, recall responses are measured by testingex-vivo proliferation and cytokine secretion in response to p31 peptide.

C1ORF32 protein fragments and/or fusion proteins thereof prevent orreduce disease onset or the severity thereof in the above studies.

Example 22

The Effect of any One of C1ORF32 Protein Fragments and/or FusionPROTEINS THEREOF IN LUPUS MOUSE MODELS

Study I: The lupus-prone mouse model, (NZB×NZW)F1 (B/W) is used.Cyclophosphamide (CTX) is the primary drug used for diffuseproliferative glomerulonephritis in patients with renal lupus, Daikh andWofsy reported that combination treatment with CTX and CTLA4-Ig was moreeffective than either agent alone in reducing renal disease andprolonging survival of NZB/NZW Fl lupus mice with advanced nephritis(Daikh and Wofsy, J Immunol, 166(5):2913-6 (2001)). In theproof-of-concept study, treatments with any one of C1ORF32 proteinfragments and/or fusion proteins thereof and CTX either alone or incombination are tested.

Blood samples are collected 3 days before the protein treatment and thenevery other week during and after treatments for plasma anti-dsDNAautoantibody analysis by ELISA. Glomerulonephritis is evaluated byhistological analysis of kidneys. Proteinuria is measured by testingfresh urine samples using urinalysis dipsticks.

C1ORF32 protein fragments and/or fusion proteins thereof have abeneficial effect in at least ameliorating lupus nephtiris.

Study II: The NZM2410-derived B6.Sle1.S1e2.S1e3 mouse model of SLE isused. NZM2410 is a recombinant inbred strain produced from NZB and NZWthat develops a highly penetrant lupus-like disease with an earlieronset of disease (Blenman et al 2006 Lab. Invest. 86: 1136-1148). Theeffect of any one of C1ORF32 protein fragments and/or fusion proteinsthereof is studied in this model by assessment of proteinuria andautoantibodies as described above.

Study III: An induced lupus model is used. This model is based onchronic graft-vs-host (cGVH) disease induced by the transfer ofIa-incompatible spleen cells from one normal mouse strain (such asB6.C-H2(bm12)/KhEg (bm12)) to another (such as C57BL/6), which causes anautoimmune syndrome resembling systemic lupus erythematosus (SLE),including anti-double-stranded DNA (anti-dsDNA) autoantibodies andimmune complex-type proliferative glomerulonephritis (Appleby et alClin. Exp. Immunol. 1989 78: 449-453); Eisenberg and Choudhury 2004Methods Mol. Med. 102:273-284).

Lupus is induced in this model following injection of spleen cells frombm12 mice into C57BL/6 recipients. The effect any one of C1ORF32 proteinfragments and/or fusion proteins thereof is studied in this model byassessment of proteinuria and autoantibodies as described above. T celland responses B cell responses will also be evaluated.

Study IV: The MRL/lpr lupus prone mouse model is used. The effect of anyone of C1ORF32 protein fragments and/or fusion proteins thereof isstudied in this model by assessment of proteinuria and autoantibodies asdescribed above.

Example 23

The Effect of any of C1ORF32 Protein Fragments and/or Fusion ProteinsThereof in the Control of Intestinal Inflammation.

An adoptive transfer mouse model of colitis in mice is used, wherebyTransfer of CD45RB^(high)-CD4⁺ naïve T cells from BALB/c mice tosyngeneic SCID mice leads to the development of an IBD-like syndrome by6-10 wks after T cell reconstitution, similar to human Crohn's disease.

SCID mice are reconstituted by i.p. injection of syngeneicCD45RB^(high)-CD4⁺ T cells either alone or cotransferred with syngeneicCD45RB^(low)-CD4⁺ or CD25⁺CD4⁺ cells (4×10⁵/mouse of each cellpopulation) (Liu et al., J Immunol. 2001; 167(3): 1830-8). Colitic SCIDmice, reconstituted with syngeneic CD45RB^(high)CD4⁺ T cells from spleenof normal mice, are treated i.p. with any one of C1ORF32 proteinfragments and/or fusion proteins thereof or Ig isotype control, twice aweek starting at the beginning of T cell transfer up to 8 wk. All miceare monitored weekly for weight, soft stool or diarrhea, and rectalprolapse. All mice are sacrificed 8 wk after T cell transfer or whenthey exhibit a loss of. 20% of original body weight. Colonic tissues arecollected for histologic and cytologic examinations. C1ORF32 proteinfragments and/or fusion proteins thereof have a beneficial effect in atleast ameliorating inflammatory bowel disease.

Example 24

The Effect of any One of C1ORF32 Protein Fragments and/or FusionProteins Thereof in Mouse Model of Psoriasis

Study I: Establishment of Psoriasis SCID Xenograft Model.

Human psoriasis plaques are transplanted on to the SCID mice. Shavebiopsies (2.5_2.5 cm) are taken from patients with generalized plaquepsoriasis involving 5-10% of the total skin that did not receive anysystemic treatment for psoriasis or phototherapy for 6 months and didnot receive any topical preparations other than emollients for 6 weeks.The biopsies are obtained from active plaques located on the thigh orarm. Each piece of biopsy is divided into four equal parts ofapproximately 1 cm2 size. Each piece is transplanted to a separatemouse.

Under general anesthesia, a graft bed of approximately 1 cm2 is createdon the shaved area of the back of a 7- to 8-week-old CB17 SCID mouse byremoving a full-thickness skin sample, keeping the vessel plexus intacton the fascia covering the underlying back muscles. The partialthickness human skin obtained by shave biopsy is then orthotopicallytransferred onto the graft bed. Nexaband, a liquid veterinary bandage(Veterinary Products Laboratories, Phoenix, Ariz.) is used to attach thehuman skin to the mouse skin and an antibiotic ointment (bacitracin) isapplied. Mice are treated intraperitoneally three times per week for 4weeks with any one of C1ORF32 protein fragments and/or fusion proteinsthereof, isotype control or CTLA4-Ig (positive control).

Punch biopsies (2 mm) are obtained on day 0 (before treatment) and day28 (after treatment) of the study period. Biopsies are snap frozen andcryosections for histopathological and immunohistochemical studies.Therapeutic efficacy is determined by comparing pre- and post treatmentdata: (i) rete peg lengths to determine the effect on epidermalthickness and (ii) the level of lymphomononuclear cell infiltrates todetermine the effect on inflammatory cellular infiltrates. (Raychaudhuriet al. 2008, J Invest Dermatol.; 128(8):1969-76; Boehncke et al., 1999Arch Dermatol Res 291:104-6).

C1ORF32 protein fragments and/or fusion proteins thereof have abeneficial effect in at least ameliorating psoriasis.

Study II: The Effect of any One of C1ORF32 Protein Fragments and/orFusion Proteins Thereof in Psoriasis and Colitis Model by AdoptiveTransfer of CD45RB_(HI) Cd4+ T Cells in SCID Mice

Immunocompromised mice are injected intraveneously (i.v.) with 0.3_10⁶CD4+CD45RBhi cells. On the day following the adoptive transfer of cells,mice are injected intraperitoneally (i.p.) with 10 microg ofstaphylococcal enterotoxin B (Davenport et al., Int Immunopharmacol.2002 April; 2(5):653-72). Recipient mice are treated with any one ofC1ORF32 protein fragments and/or fusion proteins thereof, isotypecontrol or CTLA4-Ig (positive control). Mice are evaluated once a weekfor 8 weeks for weight loss and presence of skin lesions.

Example 25

The Effect of any One of C1ORF32 Protein Fragments and/or FusionProteins Thereof in Modulating Transplant Rejection.

Study I: THE EFFECT OF any One of C1ORF32 Protein Fragments and/orFusion Proteins Thereof in a Model of Allogeneic Islet Transplantationin Diabetic Mice.

To test the effect of any one of C1ORF32 protein fragments and/or fusionproteins thereof on transplant rejection, a model of allogeneic islettransplantation is used. Diabetes is induced in C57BL/6 mice bytreatment with streptozotocin. Seven days later, the mice aretransplanted under the kidney capsule with pancreatic islets which areisolated from BALB/c donor mice. Recipient mice are treated with any oneof C1ORF32 protein fragments and/or fusion proteins thereof or withmIgG2a as a negative control. Tolerance with ECDI-fixed donorsplenocytes is used as the positive control for successful modulationislet graft rejection. Recipient mice are monitored for blood glucoselevels as a measure of graft acceptance/rejection (Luo et al., PNAS,Sep. 23, 2008_(—) vol. 105_no. 38_14527-14532).

Study II: the Effect of any One of c1ORF32 Protein Fragments and/orFusion Proteins Thereof in the Hya-Model of Skin Graft Rejection.

In humans and certain strains of laboratory mice, male tissue isrecognized as non-self and destroyed by the female immune system viarecognition of histocompatibility-Y chromosome encoded antigens (Hya).Male tissue destruction is thought to be accomplished by cytotoxic Tlymphocytes in a helper-dependent manner.

To test the effect of any one of C1ORF32 protein fragments and/or fusionproteins thereof on transplanatation, the Hya model system is used, inwhich female C57BL/6 mice receive tail skin grafts from male C57BL/6donors.

In this study, female C57BL/6 mice are engrafted with orthotopicsplit-thickness tail skin from age matched male C57BL/6 mice. The miceare treated with any one of C1ORF32 protein fragments and/or fusionproteins thereof, isotype control mIgG2a Immunodominant Hya-encoded CD4epitope (Dby) attached to female splenic leukocytes (Dby-SP) serve aspositive control for successful modulation of graft rejection (Martin etal., J Immunol. 2010 Sep. 15; 185(6): 3326-3336). Skin grafts are scoreddaily for edema, pigment loss and hair loss. Rejection is defined ascomplete hair loss and more than 80% pigment loss. In addition, T cellrecall responses of cells isolated from spleens and draining lymph nodesat different time points are studied in response to CD4 specific epitope(Dby), CD8 epitopes (Uty and Smcy) or irrelevant peptide (OVA 323-339)while anti CD3 stimulation is used as positive control forprolifereation and cytokine secretion.

Study III: The effect of any one of C1ORF32 protein fragments and/orfusion proteins thereof on graft rejection is studied in a murine modelof syngeneic bone marrow cells transplantation using the Hya modelsystem described above. Male hematopoietic cells expressing the CD45.1marker are transplanted to female host mice which express the CD45.2congenic marker. Female hosts are treated with any one of C1ORF32protein fragments and/or fusion proteins thereof or with isotype controlmIgG2a. The female hosts are followed over time and the presence ofCD45.1+ cells is monitored.

Example 26

The Effect of C1ORF32 Protein Fragments and/or Fusion Proteins Thereofin Treatment of Alopecia Areata.

To induce alopecia areata (AA), lesional skin from C3H/HeJ mice that haddeveloped spontaneous AA lesions is grafted onto the back ofnon-affected C3H/HeJ mice, as described previously (McElwee et al.,1998). Breifly, up to six grafts ˜1-1.5 cm in diameter are asepticallyremoved from each donor by pinch cutting. The grafts are placed insterile PBS while the recipient mice are prepared for transplantation.Recipient mice are anestized and a circular pice of skin ˜1.5 cm indiameter is aseptically removed from the dorsal anto-posterior midlineand replaced with a donor skin graft. The graft is oriented 180° fromnormal in order to have the hair growing in opposite direction to thereciepient hair for easy identification. Four to seven weeks aftergrafting the recipients develop initial hair loss. Recipient mice aretreated with any one of C1ORF32 protein fragments and/or fusion proteinsthereof, isotype control or CTLA4-Ig (positive control). Morphologicalchanges are examined and documented daily. Photographs are taken beforetreatment, and once per week during and after treatment. Aftercompletion of treatment, all mice are anesthesized, their dorsal skin isshaved and animals are sacrificed by cervical dislocation. Skin samplesare taken for histopathological examination and immunohistochemistry.

It will be appreciated that various features of the invention which are,for clarity, described in the contexts of separate embodiments may alsobe provided in combination in a single embodiment. Conversely, variousfeatures of the invention which are, for brevity, described in thecontext of a single embodiment may also be provided separately or in anysuitable sub-combination. It will also be appreciated by persons skilledin the art that the present invention is not limited by what has beenparticularly shown and described hereinabove.

What is claimed is:
 1. An isolated polypeptide, comprising an amino acidsequence of C1ORF32 IgV domain fragment, set forth in any one of SEQ IDNOs: 29, 30, 41-105, with the proviso that the amino acid sequence doesnot include the complete, exact sequence of SEQ ID NO: 35 or SEQ IDNO:36.
 2. The isolated peptide of claim 1, wherein the isolatedpolypeptide has less than 90% identity with SEQ ID NO:35 or SEQ IDNO:36.
 3. The isolated peptide of claim 2, wherein the isolated peptidehas less than 85% identity with SEQ ID NO:35 or SEQ ID NO:36.
 4. Theisolated peptide of claim 3, wherein the isolated peptide has less than80% identity with SEQ ID NO:35 or SEQ ID NO:36.
 5. The isolated peptideof any of claims 1-4, wherein the isolated polypeptide consistsessentially of the amino acid sequence as set forth in any one of SEQ IDNOs: 29, 30, 41-105, or optionally an amino acid sequence that is up to95% identical thereof
 6. A fusion protein comprising the polypeptideaccording to any of claims 1-5, fused to a heterologous sequence,directly or indirectly via a linker peptide, a polypeptide sequence or achemical linker.
 7. The fusion protein of 6, wherein the heterologoussequence comprises at least a portion of an immunoglobulin constantdomain.
 8. The fusion protein of claim 7, comprising an immunoglobulinheavy chain constant region corresponding to an antibody isotypeselected from the group consisting of an IgG1, IgG2, IgG3, IgG4, IgM,IgE, IgA and IgD.
 9. The fusion protein of 8, wherein the immunoglobulinconstant domain comprises the hinge, CH2 and CH3 regions of a human IgGimmunoglobulin, selected from the group consisting of Cγ1, Cγ2, Cγ3 andCγ4 chain.
 10. The fusion protein of any of claims 6-9, furthercomprising a domain that mediates dimerization or multimerization of thefusion protein to form homodimers, heterodimers, homomultimers, orheteromultimers.
 11. The fusion protein of 10, wherein the domain thatmediates dimerization or multimerization is selected from the groupconsisting of one or more cysteines that are capable of forming anintermolecular disulfide bond with a cysteine on the partner fusionprotein, a coiled-coil domain, an acid patch, a zinc finger domain, acalcium hand domain, a CHI region, a CL region, a leucine zipper domain,an SH2 (src homology 2) domain, an SH3 (src Homology 3) domain, a PTB(phosphotyrosine binding) domain, a WW domain, a PDZ domain, a 14-3-3domain, a WD40 domain, an EH domain, a Lim domain, an isoleucine zipperdomain, and a dimerization domain of a receptor dimer pair.
 12. Thefusion protein of any of claims 6-11, comprising a polypeptide having anamino acid sequence set forth in any one of SEQ ID NOs: 20, 21, 31 or115.
 13. The fusion protein of claim 12, wherein said fusion proteincomprises the amino acid sequence set forth in anyone of SEQ ID NOs: 29,30, 41-105, fused to human IgG1 Fc set forth in any one of SEQ ID NOs:20, 21, or
 115. 14. The fusion protein of claim 13, wherein the aminoacid sequence of said fusion protein is set forth in SEQ ID NO: 39,108-112, 116-190.
 15. The fusion protein of claim 14, wherein the aminoacid sequence is set forth in any of SEQ ID NOs:112, 120 oralternatively in any of SEQ ID NOs:110,
 136. 16. A dimeric proteincomprising a first and a second fusion protein, wherein the first andthe second fusion proteins comprise the fusion protein of any of claims6-13, wherein the first and the second fusion proteins are bound to oneanother by covalent or noncovalent bonds to form a dimer.
 17. Apharmaceutical composition comprising a protein of any of claims 1-5 ora fusion protein of any of claims 6-16, and a pharmaceuticallyacceptable diluent or carrier, adapted for treatment of any immunerelated disorder and infectious disorder.
 18. A use of a protein of anyof claims 1-5 or a fusion protein of any of claims 6-16 orpharmaceutical composition according to claim 20 for treatment of any ofimmune related disorder and infectious disorder.
 19. The use accordingto claim 18, wherein the polypeptide is attached to a detectable ortherapeutic moiety.
 20. A method for treating an immune related disorderor an infectious disorder in a subject in need thereof, comprisingadministering to the subject an effective amount of the polypeptide ofany of claims 1-5 or fusion protein of any of claims 6-16 orpharmaceutical composition according to claim
 17. 21. The method of 20,wherein administering an effective amount of the polypeptide of any ofclaims 1-5 or fusion protein of any of claims 6-16 or pharmaceuticalcomposition according to claim 20 to the subject inhibits or reducesdifferentiation of, proliferation of, activity of, and/or cytokineproduction and/or secretion by an immune cell selected from the groupconsisting of Th1, Th17, and/or Th22, other cells that secrete, or cellsthat cause other cells to secrete, inflammatory molecules.
 22. Themethod of 20, wherein the polypeptide of any of claims 1-5 or fusionprotein of any of claims 6-16 or pharmaceutical composition according toclaim 17 is administered in an effective amount to inhibit or reducedifferentiation of, proliferation of, activity of, and/or cytokineproduction and/or secretion by Th1, Th17 and/or Th22 cells.
 23. Themethod of claim 20, wherein the polypeptide of any of claims 1-5 orfusion protein of any of claims 6-16 or pharmaceutical compositionaccording to claim 17 is administered in an effective amount to enhancethe suppressive or immunomodulatory effect of Tregs and/or Th2 cells onTh1 or Th17 cells.
 24. The method of claim 20, wherein the polypeptideof any of claims 1-5 or fusion protein of any of claims 6-16 orpharmaceutical composition according to claim 17 is administered in aneffective amount to promote or enhance IL-10 production.
 25. The methodof claim 20, wherein the polypeptide of any of claims 1-5 or fusionprotein of any of claims 6-16 or pharmaceutical composition according toclaim 17 is administered in an effective amount to increase cell numbersor increase populations of any of Tregs and/or Th2 cells.
 26. The methodof claim 20, wherein the polypeptide of any of claims 1-5 or fusionprotein of any of claims 6-16 or pharmaceutical composition according toclaim 17 is administered in an effective amount to inhibit the Th1and/or Th17 pathways and to enhance the activity of Tregs and/or Th2cells on the Th1 and Th17 pathways and/or to promote or enhance IL-10secretion.
 27. The method of claim 20, wherein the polypeptide of any ofclaims 1-5 or fusion protein of any of claims 6-16 or pharmaceuticalcomposition according to claim 17 is administered in an effective amountfor reducing proinflammatory molecule production in a subject.
 28. Themethod according to any one of claims 20-27 further comprisingadministering a second therapeutic agent effective for treatment of anyimmune related disorder and infectious disorder.
 29. The C1ORF32polypeptide, fusion protein, pharmaceutical composition, method, or useaccording to any of the above claims, wherein the immune relateddisorder is selected from the group consisting of autoimmune disease andimmune disorder associated with graft transplantation rejection, andwherein the infectious disorder is selected from the disease caused bybacterial infection, viral infection, fungal infection and/or otherparasite infection.
 30. The C1ORF32 polypeptide, fusion protein,pharmaceutical composition, method, or use according to claim 29,wherein the immune disorder associated with graft transplantationrejection is selected from the group consisting of acute and chronicrejection of organ transplantation, allogeneic stem celltransplantation, autologous stem cell transplantation, bone marrowtransplantation, and graft versus host disease.
 31. The C1ORF32polypeptide, fusion protein, pharmaceutical composition, method, or useaccording to claim 29, wherein the autoimmune disease is selected fromthe group consisting of multiple sclerosis, rheumatoid arthritis;psoriatic arthritis, discoid lupus erythematosus, systemic lupuserythematosus (SLE); ulcerative colitis; Crohn's disease; benignlymphocytic angiitis, autoimmune lymphoproliferative syndrome,sarcoidosis, autoimmune thrombocytopenic purpura, idiopathicthrombocytopenic purpura, pure red cell aplasia, Sjogren's syndrome,rheumatic disease, polymyalgia rheumatica, mixed connective tissuedisease, inflammatory rheumatism, degenerative rheumatism,extra-articular rheumatism, juvenile arthritis, juvenile rheumatoidarthritis, systemic juvenile idiopathic arthritis, arthritis uratica,muscular rheumatism, chronic polyarthritis, reactive arthritis, Reiter'ssyndrome, rheumatic fever, relapsing polychondritis, Raynaud'sphenomenon, vasculitis, cryoglobulinemic vasculitis, ANCA-associatedvasculitis, temporal arteritis, giant cell arteritis, Takayasuarteritis, Behcet's disease, antiphospholipid syndrome, myastheniagravis, autoimmune haemolytic anaemia, Guillain-Barre syndrome, chronicimmune polyneuropathy, chronic inflammatory demyelinatingpolyneuropathy, autoimmune thyroiditis, insulin dependent diabetesmellitus, type I diabetes, Addison's disease, membranousglomerulonephropathy, polyglandular autoimmune syndromes, Goodpasture'sdisease, autoimmune gastritis, autoimmune atrophic gastritis, perniciousanaemia, pemphigus, pemphigus vulgaris, cirrhosis, primary biliarycirrhosis, idiopathic pulmonary fibrosis, myositis, dermatomyositis,juvenile dermatomyositis, polymyositis, fibromyositis, myogelosis,celiac disease, celiac sprue dermatitis, immunoglobulin A nephropathy,Henoch-Schonlein purpura, Evans syndrome, atopic dermatitis, psoriasis,psoriasis vulgaris, psoriasis arthropathica, Graves' disease, Graves'ophthalmopathy, scleroderma, systemic scleroderma, progressive systemicscleroderma, diffuse scleroderma, localized scleroderma, Crest syndrome,asthma, allergic asthma, allergy, primary biliary cirrhosis, Hashimoto'sthyroiditis, fibromyalgia, chronic fatigue and immune dysfunctionsyndrome (CFIDS), primary myxedema, sympathetic ophthalmia, autoimmuneinner ear disease, autoimmune uveitis, autoimmune chronic activehepatitis, collagen diseases, ankylosing spondylitis, periarthritishumeroscapularis, panarteritis nodosa, polyarteritis nodosa,chondrocalcinosis, Wegener's granulomatosis, microscopic polyangiitis,chronic urticaria, bullous skin disorders, pemphigoid, bullouspemphigoid, cicatricial pemphigoid, vitiligo, atopic eczema, eczema,chronic urticaria, autoimmune urticaria, normocomplementemic urticarialvasculitis, hypocomplementemic urticarial vasculitis, alopecia areata,alopecia universalis, alopecia totalis, Devic's disease, perniciousanemia, childhood autoimmune hemolytic anemia, idiopathic autoimmunehemolytic anemia, refractory or chronic Autoimmune Cytopenias,Prevention of development of Autoimmune Anti-Factor VIII Antibodies inAcquired Hemophilia A, Cold agglutinin disease, Neuromyelitis Optica,Stiff Person Syndrome, gingivitis, periodontitis, pancreatitis,myocarditis, gastritis, gout, gouty arthritis, idiopathic pericarditis,anti-synthetase syndrome, scleritis, macrophage activation syndrome,PAPA Syndrome, Blau's Syndrome, adult and juvenile Still's disease,cryopyrin associated periodic syndrome, Muckle-Wells syndrome, familialcold auto-inflammatory syndrome, neonatal onset multisystem inflammatorydisease, chronic infantile neurologic cutaneous and articular syndrome,familial Mediterranean fever, Hyper IgD syndrome, Schnitzler's syndrome,autoimmune retinopathy, age-related macular degeneration, and TNFreceptor-associated periodic syndrome (TRAPS).
 32. The C1ORF32polypeptide, fusion protein, pharmaceutical composition, method, or useaccording to claim 29, wherein the autoimmune disease is selected fromthe group consisting of multiple sclerosis, rheumatoid arthritis, type Idiabetes, psoriasis, systemic lupus erythematosus, inflammatory Bowel'sdisease, uveitis, and Sjogren's syndrome.
 33. The C1ORF32 polypeptide,fusion protein, pharmaceutical composition, method, or use according toclaim 32, wherein the multiple sclerosis is selected from the groupconsisting of benign multiple sclerosis, relapsing remitting multiplesclerosis, secondary progressive multiple sclerosis, primary progressivemultiple sclerosis, chronic progressive multiple sclerosis,transitional/progressive multiple sclerosis, progressive relapsingmultiple sclerosis, rapidly worsening multiple sclerosis,clinically-definite multiple sclerosis, malignant multiple sclerosis,also known as Marburg's Variant, acute multiple sclerosis and conditionrelating to multiple sclerosis, selected from the group consisting ofDevic's disease, also known as Neuromyelitis Optica; acute disseminatedencephalomyelitis, acute demyelinating optic neuritis, demyelinativetransverse myelitis, Miller-Fisher syndrome,encephalomyelradiculoneuropathy, acute demyelinative polyneuropathy,tumefactive multiple sclerosis and Balo's concentric sclerosis.
 34. TheC1ORF32 polypeptide, fusion protein, pharmaceutical composition, method,or use according to claim 32, wherein the rheumatoid arthritis isselected from the group consisting of rheumatoid arthritis, gout andpseudo-gout, juvenile idiopathic arthritis, juvenile rheumatoidarthritis, Still's disease, ankylosing spondylitis, rheumatoidvasculitis, and conditions relating to rheumatoid arthritis, selectedfrom the group consisting of osteoarthritis, sarcoidosis,Henoch-Schönlein purpura, Psoriatic arthritis, Reactive arthritis,Spondyloarthropathy, septic arthritis, HaemochromatosisHepatitis,vasculitis, Wegener's granulomatosis, Lyme disease, FamilialMediterranean fever, Hyperimmunoglobulinemia D with recurrent fever, TNFreceptor associated periodic syndrome, and Enteropathic arthritisassociated with inflammatory bowel disease.
 35. The C1ORF32 polypeptide,fusion protein, pharmaceutical composition, method, or use according toclaim 32, wherein the uveitis is selected from the group consisting ofanterior uveitis (or iridocyclitis), intermediate uveitis (parsplanitis), posterior uveitis (or chorioretinitis) and the panuveiticform.
 36. The C1ORF32 polypeptide, fusion protein, pharmaceuticalcomposition, method, or use according to claim 32, wherein theinflammatory bowel disease is selected from the group consisting ofCrohn's disease and ulcerative colitis (UC) and condition relating toIBD selected from the group consisting of Collagenous colitis,Lymphocytic colitis, Ischaemic colitis, Diversion colitis, Behçet'sdisease, Indeterminate colitis.
 37. The C1ORF32 polypeptide, fusionprotein, pharmaceutical composition, method, or use according to claim32, wherein the psoriasis is selected from the group consisting ofNonpustular Psoriasis including Psoriasis vulgaris and Psoriaticerythroderma (erythrodermic psoriasis), Pustular psoriasis includingGeneralized pustular psoriasis (pustular psoriasis of von Zumbusch),Pustulosis palmaris et plantaris (persistent palmoplantar pustulosis,pustular psoriasis of the Barber type, pustular psoriasis of theextremities), Annular pustular psoriasis, Acrodermatitis continua,Impetigo herpetiformis. Optionally, conditions relating to psoriasisinclude, e.g., drug-induced psoriasis, Inverse psoriasis, Napkinpsoriasis, Seborrheic-like psoriasis, Guttate psoriasis, Nail psoriasis,Psoriatic arthritis.
 38. The C1ORF32 polypeptide, fusion protein,pharmaceutical composition, method, or use according to claim 32,wherein the diabetes is selected from the group consisting ofinsulin-dependent diabetes mellitus, idiopathic diabetes, juvenile type1 diabetes, maturity onset diabetes of the young, latent autoimmunediabetes in adults, gestational diabetes, and condition relating to type1 diabetes selected from the group consisting of neuropathy includingpolyneuropathy, mononeuropathy, peripheral neuropathy andautonomicneuropathy; eye complications: glaucoma, cataracts,retinopathy.
 39. The C1ORF32 polypeptide, fusion protein, pharmaceuticalcomposition, method, or use according to claim 32, wherein the Sjogren'ssyndrome is selected from the group consisting of Primary Sjogren'ssyndrome and Secondary Sjogren's syndrome and condition relating toSjogren's syndrome selected from the group consisting of connectivetissue disease, such as rheumatoid arthritis, systemic lupuserythematosus, or scleroderma. Other complications include pneumonia,polmunary fibrosis, interstitial nephritis, inflammation of the tissuearound the kidney's filters, glomerulonephritis, renal tubular acidosis,carpal tunnel syndrome, peripheral neuropathy, cranial neuropathy,primary biliary cirrhosis (PBC), cirrhosis, Inflammation in theesophagus, stomach, pancreas, and liver (including hepatitis),Polymyositis, Raynaud's phenomenon, Vasculitis, Autoimmune thyroidproblems, lymphoma.
 40. The C1ORF32 polypeptide, fusion protein,pharmaceutical composition, method, or use according to claim 32,wherein the systemic lupus erythematosus is selected from the groupconsisting of discoid lupus, lupus arthritis, lupus pneumonitis, lupusnephritis, and condition relating to systemic lupus erythematosus,selected from the group consisting of osteoarticular tuberculosis,antiphospholipid antibody syndrome, inflammation of various parts of theheart, such as pericarditis, myocarditis, and endocarditis, Lung andpleura inflammation, pleuritis, pleural effusion, chronic diffuseinterstitial lung disease, pulmonary hypertension, pulmonary emboli,pulmonary hemorrhage, and shrinking lung syndrome, lupus headache,Guillain-Barré syndrome, aseptic meningitis, demyelinating syndrome,mononeuropathy, mononeuritis multiplex, myasthenia gravis, myelopathy,cranial neuropathy, polyneuropathy, vasculitis.
 41. The C1ORF32polypeptide, fusion protein, pharmaceutical composition, method, or useaccording to claim 32, wherein treating comprises one or more ofpreventing, curing, managing, reversing, attenuating, alleviating,minimizing, suppressing, managing, or halting the deleterious effects ofthe above-described diseases.
 42. The C1ORF32 polypeptide, fusionprotein, pharmaceutical composition, method, or use according to claim41, wherein the treating comprises treatment of immune related disorderwithout global immunosuppression.
 43. The C1ORF32 polypeptide, fusionprotein, pharmaceutical composition, method, or use according to claim41, wherein the treatment of immune related disorder comprises inductionof immune tolerance.
 44. The C1ORF32 polypeptide, fusion protein,pharmaceutical composition, method, or use according to claim 41,wherein the treatment and/or prevention comprises inhibition ofinfiltration of reactive T lymphocytes into the central nervous system.45. The C1ORF32 polypeptide, fusion protein, pharmaceutical composition,method, or use according to claim 41, wherein the treatment and/orprevention comprises prevention of damage to the myelin coat of neuralcells in the central nervous system.
 46. The C1ORF32 polypeptide, fusionprotein, pharmaceutical composition, method, or use according to claim41, wherein treatment comprises reducing the severity of the disease,reducing the frequency of episodes of the disease, reducing the durationof such episodes, or reducing the severity of such episodes or acombination thereof.
 47. A method for selecting a subject for treatmentwith a C1ORF32 fusion protein comprising screening subjects for levelsof one or more cytokines selected from the group consisting of IL-1beta,TNF-alpha, TGF-beta, IFN-gamma, IL-10, IL-17, IL-6, IL-23, IL-22, andIL-21, or MMPs, comparing the levels of the cytokines to the levels ofcytokines or MMPs in a control subject that does not have an immunerelated disorder, and administering to the subject an effective amountof the polypeptide of any of claims 1-5 or fusion protein of any ofclaims 6-16 or pharmaceutical composition according to claim 17 toinhibit or reduce one or more symptoms of the immune related disorder ifthe levels of one or more cytokines are elevated in the subject comparedto levels in the control subject that does not have the immune relateddisorder.
 48. A method for selecting a subject for treatment with aC1ORF32 fusion protein comprising screening subjects for levels of mRNAencoding one or more cytokines selected from the group consisting ofIL-1beta, TNF-alpha, TGF-beta, IFN-gamma, IL-10, IL-17, IL-6, IL-23,IL-22, and IL-21, or MMPs, comparing the levels of the mRNAs encodingcytokines or MMPs to levels of the mRNAs encoding cytokines or MMPs in acontrol subject that does not have an inflammatory disorder, andadministering to the subject an effective amount of the polypeptide ofany of claims 1-5 or fusion protein of any of claims 6-16 orpharmaceutical composition according to claim 17 to inhibit or reduceone or more symptoms of an immune related disorder if the levels of oneor more mRNAs encoding cytokines are elevated in the subject compared tolevels in the control subject that does not have an immune relateddisorder.
 49. The method of any of the above claims, wherein the subjectdid not previously respond to treatment with TNF blockers.
 50. A methodfor combining therapeutic vaccination with an antigen along withadministration of any one of the polypeptide of any of claims 1-5 orfusion protein of any of claims 6-16 or pharmaceutical compositionaccording to claim 17, for treatment of infection.
 51. A method forcombining of any one of the polypeptide of any of claims 1-5 or fusionprotein of any of claims 6-16 or pharmaceutical composition according toclaim 17, used as adjuvant, along with an antigen in a vaccine, in orderto increase the immune response.
 52. The method of claim 50 or 51,wherein the antigen is a viral antigen, bacterial antigen, fungalantigen, parasite antigen, and/or other pathogen's antigen.